BACKGROUND AND OBJECTIVESData on HIV drug resistance in Madagascar are rare and outdated. In this study, we assessed the prevalence of HIV drug resistance mutations to antiretrovirals (ARVs) and genetic diversity of circulating strains in treatment-naive people living with HIV (PLHIV) in Madagascar.MATERIALS AND METHODSWe amplified the protease (PR), fragments of the Reverse Transcriptase (RT) and Integrase (IN) genes according to the French ANRS protocol. The amplicons were sequenced using next-generation sequencing technology on an Illumina platform (MiSeq). We determined HIV-1 subtypes through phylogenetic analysis using maximum likelihood in PhyML. Resistance interpretation was performed using the Stanford algorithm (version 9.5.1).RESULTSWe included 239 HIV-infected adults and children, sampled between January 2019 and November 2023, with a median age of 30 years and a mean plasma HIV viral load of 6.3 Log copies/mL. We sequenced at least one genomic fragment (PR or RT or IN) of the 239 samples, but 9 were excluded from analysis (mean depth < 10,000×). Phylogenetic analysis of 230 sequences revealed the presence of subtype C (33.91 %), A1 (11.30 %), B (11.30 %), CRF02_AG (9.56 %), subtype G (3.04 %), subtype D (0.43 %), CRF01_AE (0.43 %), and a significant proportion of unique recombinant forms (URFs) (30.30 %). The prevalence of transmitted drug resistance (TDR) was 4.95 % (10/202) among patients aged 15 years and older. When stratified by ARV class, this prevalence was 4.79 % for non-nucleoside reverse transcriptase inhibitors (NNRTIs), 0.59 % for Nucleoside Reverse Transcriptase inhibitors (NRTIs), and 0.50 % for integrase strand transfer inhibitors (INSTIs). Among children under 15 years old (n = 28), the prevalence of TDR was 14.28 % (4/28), with all mutations conferring resistance to NNRTIs. No mutation conferring resistance to protease inhibitors was found, neither in children nor in adults.CONCLUSIONOur results show a low prevalence of ARV resistance mutations among adult treatment-naive PLHIV in Madagascar. In children under 15 years old, 92 % were infants under two years old, the high resistance rate is likely related to mother-to-child transmission. No resistance mutation to dolutegravir was detected. We also observed high frequencies of subtypes C, B, A1 and a high proportion of URFs, highlighting an ongoing dynamic epidemic.