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Purpose: Non-tuberculous mycobacteria (NTM) are emerging pathogens with increasing clinical significance worldwide, yet regional epidemiological and drug susceptibility data remain limited. The purpose of this study is to analyze the distribution, demographic characteristics, and drug susceptibility of NTM infections in Nanjing, and to provide a basis for clinical diagnosis and treatment. Patients and Methods: Confirmed cases of NTM infection were collected from the Tuberculosis Department of Nanjing Second Hospital between January 2023 and November 2025. Clinical isolates were cultured using the BACTEC MGIT960 system. Mycobacterium tuberculosis was ruled out by MPB64 antigen detection (immunochromatographic assay). Species identification was performed using either PCR-reverse dot blot hybridization or nanopore sequencing. Subsequently, in vitro drug susceptibility testing was conducted via the broth microdilution method. Results: Among 249 cases of NTM detected, the most common bacterial strains are Mycobacterium intracellulare ( M. intracellulare , 105/249, 42.2%) and Mycobacterium avium ( M. avium , 82/249, 32.9%), with Mycobacterium abscessus accounting for 8.4% ( M. abscessus , 21/249, 8.4%), 26 cases (26/249, 10.4%) were NTM mixed infection. The most common mixed infection pattern is M. abscessus and M. avium complex (MAB and MAC, 10/26, 38.5%). The age distribution of patients is mainly middle-aged and elderly, with females being the majority. The drug susceptibility results showed that amikacin (AMK) had a susceptibility rate of ≥ 93.3% to most strains, but only 61.9% to M. abscessus ; The susceptibility rate of clarithromycin (CLR) to M. abscessus was 47.6%; moxifloxacin (MFX) has a susceptibility rate of over 97% to M. intracellulare and M. avium . In contrast, susceptibility to sulfamethoxazole (SMZ), imipenem/cilastatin (I/C), minocycline (MH), and doxycycline (DOX) was low, with rates below 50% for most species and 0% for I/C and MH across all isolates. Mixed infections exhibited distinct susceptibility profiles compared to single species. For example, M. abscessus -MAC mixed infections retained high susceptibility to MFX (90%) and AMK (90%), whereas M. abscessus-Mycobacterium chelonae mixed infection showed high macrolide resistance (> 80%) but 80% susceptibility to linezolid (LZD). Conclusion: This study reveals distribution of NTM species in Nanjing Jiangsu Province China and distinct antimicrobial susceptibility profiles between single and mixed NTM infections, highlighting the clinical complexity of NTM disease, reinforce the necessity of species-level identification and antimicrobial susceptibility testing to guide individualized treatment and address the growing challenge of antimicrobial resistance in clinical practice. Keywords: NTM mixed infection, minimum inhibitory concentration, MIC, antimicrobial susceptibility, antimicrobial resistance