BACKGROUND: After the rapid scale-up of antiretroviral therapy (ART) in resource-limited settings, surveillance of primary drug resistance mutations (DRMs) among ART-naive individuals has important public health benefits. Although a highly successful national ART programme initiated by the Government of India exists, data on the prevalence of primary DRMs is scarce. The objective of the study is to estimate the prevalence, pattern and spectrum of population-based primary DRMs in therapy-naive HIV-1-infected individuals using clinical strains and database sequences from seven HIV prevalent states of India. METHODS: Drug resistance genotyping was performed on either plasma RNA or whole-blood genomic DNA using a validated in-house method on 170 HIV-1-positive therapy-naive individuals. An additional 679 database-derived sequences from four other states were included in the analysis. The WHO-recommended list of mutations (SDRM_2009) for nucleoside reverse transcriptase inhibitors (NRTIs) and non-nucleoside reverse transcriptase inhibitors (NNRTIs) were used for interpretation of DRMs. Trends of primary DRMs before and after the ART rollout were studied. RESULTS: The overall prevalence of primary DRMs was 2.6% in the selected states of India when clinical isolates as well as database-derived sequences were combined. Common mutations included T69D and D67N (NRTI mutations), and L100I, K101E, K103N and Y181C (NNRTI mutations). There was a significant increase in NNRTI mutations over time. CONCLUSIONS: The overall DRM prevalence in this study was low. However, an increasing trend in primary NNRTI resistance has been observed during the past decade. Establishment of HIV drug resistance threshold surveillance will be useful in understanding further trends of transmitted resistance.