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Abstract India carries a high burden of neglected tropical diseases (NTDs), yet the extent to which essential diagnostic services align with local disease burden across public health facilities remains unclear. We conducted a cross-sectional assessment of diagnostic availability for major NTDs in 332 public health facilities across seven states and one union territory, including sub-centers, primary health centers, community health centers, and district hospitals. Diagnostic availability for malaria, dengue, Japanese encephalitis, chikungunya, lymphatic Filariasis, Leishmaniasis, helminthic infections, and HIV was evaluated using the ICMR National Essential Diagnostics List (2019), and a Diagnostic Readiness Index (DRI) was calculated at the facility, district, and state levels. Big three diseases like tuberculosis, malaria, and HIV are not neglected anymore in terms of funding and research, but their interaction with classic NTDs highlights how these diseases, though different in classification, create a dual burden in vulnerable populations, like in India. Diagnostic readiness increased with higher levels of care but showed limited concordance with disease burden. Malaria diagnostics were widely available across all tiers (mean district DRI: 84.34%), reflecting sustained programmatic prioritization. In contrast, diagnostic availability for dengue (40.36%), lymphatic Filariasis (29.22%), helminthic infections (25.30%), Japanese encephalitis (8.13%), and Leishmaniasis (5.72%) remained low, including in districts reporting substantial disease burden. The greatest mismatch between burden and diagnostic availability was observed at sub-centers and primary health centers, whereas district hospitals showed a more favorable alignment. These findings indicate that essential diagnostic deployment for NTDs in India remains uneven and weakly responsive to epidemiological need. Strengthening burden-informed, decentralized access to point-of-care diagnostics—particularly at peripheral and primary care levels—is critical to improve early case detection, surveillance accuracy, and progress toward national NTD control and elimination targets. Author Summary Neglected tropical diseases (NTDs) continue to affect millions of people in India, particularly those living in underserved and resource-limited settings. Early and accurate diagnosis is essential for timely treatment, surveillance, and control; however, the availability of essential diagnostic services across different levels of the public health system remains poorly understood. In this study, we assessed the availability of diagnostics for major NTDs across 332 public health facilities, including sub-centers, primary health centers, community health centers, and district hospitals in seven states and one union territory, using the ICMR National Essential Diagnostics List (2019) and a Diagnostic Readiness Index. We found that diagnostic readiness generally increased with higher levels of care but showed limited alignment with local disease burden. Malaria diagnostics were widely available, reflecting sustained programmatic prioritization, whereas diagnostics for dengue, lymphatic filariasis, helminthic infections, Japanese encephalitis, and leishmaniasis were markedly limited, even in areas with substantial reported burden. The largest gaps were observed at peripheral and primary care facilities, which are often the first point of contact for vulnerable populations. Our findings highlight a critical mismatch between disease burden and diagnostic deployment for NTDs in India. Strengthening decentralized, burden-informed access to point-of-care diagnostics—especially at sub-centers and primary health centers—could improve early case detection, enhance surveillance accuracy, and support more effective control and elimination efforts. This study provides policy-relevant evidence to guide health system strengthening and equitable diagnostic access for NTD-endemic regions.