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Abstract Background: The long-term bio-distribution and persistence of components from COVID-19 RNA vaccines remain insufficiently characterized. Emerging evidence suggests that prolonged spike protein expression, residual RNA, and plasmid DNA fragments may contribute to multi-system post-vaccination syndromes. Case Presentation: We report a 55-year-old male who received three doses of the Pfizer–BioNTech COVID-19 mRNA vaccine and subsequently developed progressive multi-organ dysfunction consistent with post-COVID-19 vaccine syndrome (PCVS), involving cardiopulmonary, neurologic, musculoskeletal, gastrointestinal, autonomic, otolaryngologic, audiovestibular, immune, ophthalmic, dermatologic, and psychiatric domains. Clinical manifestations included: pulmonary emboli; delayed MRI-confirmed myocarditis; neurocognitive impairment; small fiber neuropathy; autonomic dysfunction; myalgia; chronic pancreatic and gastrointestinal involvement; worsened tinnitus with sensorineural hearing loss; voice dysphagia and dysphonia; ophthalmic disturbances; chronic dermatologic inflammation; and anxiety/depression. The case was evaluated through a uniquely extensive longitudinal, multi-domain clinical investigation spanning molecular, immunologic, genetic, proteomic, transcriptomic, and tissue-based analyses, undertaken to characterize disease mechanisms and exclude alternative etiologies. Diagnostic Evaluation: After >40 emergency department visits and >200 outpatient specialty encounters, the patient underwent >100 non-routine laboratory investigations and >100 imaging/functional studies. This evaluation systematically excluded underlying etiologic mechanisms across infectious, autoimmune, rheumatologic, endocrine, genetic, hematologic, malignant, toxic/medication-related, cardiovascular/vascular, metabolic, and primary neurologic domains. Testing remained largely nondiagnostic. A possible undocumented/undiagnosed asymptomatic infection manifesting as Long COVID was suspected after myocarditis diagnosis, and serology was pursued; unexpected results prompted expanded immune and tissue-based testing for spike- and vaccine-derived components. SARS-CoV-2 nucleocapsid antibodies were negative across five separate time points spanning 809–1,433 days post-vaccination, confirmed by three independent laboratories. The patient remains nucleocapsid negative with persistently elevated spike antibody levels (4,553 U/mL) 1,433 days after the final vaccination. Specimen Collection and Analytical Methods: Blood and skin tissue specimens were obtained at multiple time points between 852–1,364 days after the final Pfizer–BioNTech COVID-19 mRNA vaccination. Biological compartments analyzed included plasma, circulating exosomes, peripheral blood mononuclear cells (PBMCs), and skin tissue. Specimens were evaluated across multiple independent laboratories using diverse analytical methodologies, including ELISA, automated immunohistochemistry, RT-PCR, standard PCR with Sanger sequencing confirmation, whole-genome sequencing, transcriptomic profiling, and quantitative mass spectrometry. Circulating Molecular Findings: At 852 days post-vaccination, blood-based immune testing identified detectable SARS-CoV-2 S1 protein within classical and non-classical monocyte subsets with associated cytokine and immune marker abnormalities. At 1,173 days post-vaccination, high-sensitivity ELISA detected free Wuhan spike protein in plasma (129.0 ± 4.1 fg/mL) and in circulating exosomes (11.6 ± 0.1 fg/mL). At 1,284 days, RT-PCR identified vaccine-derived spike mRNA within circulating exosomes, whereas PBMC RNA remained negative following DNase-treated extraction and amplicon-specific PCR targeting three spike ORF regions (S1–S3). Serologic profiling at 1,173 and 1,284 days post-vaccination demonstrated persistently elevated spike-specific IgG4 concentrations (354.4 ± 22.4 ng/mL and 320.2 ± 4.4 ng/mL, respectively), consistent with ongoing antigenic stimulation and an immune-tolerance–skewed response. Tissue-Level Molecular and Histopathologic Findings: Serial skin biopsies at 1,160, 1,249, and 1,364 days post-vaccination, all from truncal skin within areas of clinically active Grover’s disease, were nucleocapsid negative and demonstrated persistent spike protein deposition in endothelial cells and macrophages by automated immunohistochemistry with histopathologic correlation. Spike protein was also found in nerve fibers at 1,364 days. The 1,364-day skin biopsy contained multiple plasmid DNA elements, including spike gene sequences (S1–S3), ori1/ori2, and the SV40 enhancer, confirming durable retention of vaccine-derived DNA in somatic tissue by PCR amplification with agarose gel electrophoresis and Sanger sequencing. Multi-Omic Analysis: Whole-genome sequencing structural variant analysis at 1,277 days post-vaccination revealed widespread genomic instability, with large duplications and deletions affecting EGFR, MYC, ERBB2, and ETV6/RUNX1, while RNA–DNA comparison showed RNA-only variants in ribosomal, NMD, small-RNA, epigenetic, and TP53 pathways. Transcriptomic profiling of whole blood highlighted oxidative stress, vascular activation, and nuclear fragility. Urine proteomics using quantitative mass spectrometry confirmed systemic inflammation with complement overactivation (CFH), redox imbalance (PRDX1), and sustained antibody responses, supported by risk alleles HLA-B07:02 and DRB1*11:04. Conclusion: This case documents the longest reported in vivo persistence of vaccine-derived mRNA, plasmid DNA fragments, and spike protein following mRNA vaccination, with reproducible detection across multiple independent laboratories, distinct biological compartments, and complementary molecular detection systems extending beyond 3.5 years after the final dose. Spike protein, spike mRNA sequences, and plasmid backbone elements were identified in both immune cells and somatic tissue, with continued absence of SARS-CoV-2 nucleocapsid protein or antibodies, effectively excluding prior infection as the source. The convergence of these observations across longitudinal blood and tissue sampling provides direct evidence that mRNA vaccine-derived genetic material and its translated protein products can persist in vivo for years following administration. In parallel, multi-omic analyses revealed sustained genomic instability and transcriptomic dysregulation more than 3.5 years post-vaccination, suggesting that persistent vaccine-derived material may be associated with long-term alterations in host genomic and molecular pathways. These data challenge prevailing assumptions regarding rapid degradation and short-lived biological activity of mRNA vaccine components and underscore the need for controlled longitudinal studies to determine prevalence, mechanisms, and clinical consequences of persistent vaccine-derived material.