Background: There are few data on the prevalence of acquired drug resistance mutations (ADRs) in Hunan Province, China, that could affect the effectiveness of antiretroviral therapy (ART). Objectives: The main objectives of this study were to determine the prevalence of acquired drug resistance (ADR) the epidemic characteristics of HIV-1-resistant strains among ART-failed HIV patients in Hunan Province, China. Methods: ART-experienced and virus suppression failure subjects in Hunan between 2012 and 2017 were evaluated by genotyping analysis and mutations were scored using the HIVdb.stanford.edu algorithm to infer drug susceptibility. Results: The prevalence of HIV-1 ADR were 2.76, 2.30, 2.98, 2.62, 2.23and 2.17%, respectively, from 2012 to 2017. Overall 2295 sequences were completed from 2932 ART-failure patients, and 914 of these sequences were found to have drug resistance mutation. The most common subtype was AE (64.14%), followed by BC (17.91%) and B (11.50%). Among those 914 patients with drug resistance mutations,93.11% had NNRTI-associated drug resistance mutations, 74.40% had NRTI drug resistance mutations (DRMs) and 6.89% had PI DRMs. Dual-class mutations were observed in 591 (64.66%) cases, and triple-class mutations were observed in 43 (4.70%) cases. M184V (62.04%), K103N (41.90%) and I54L (3.83%) were the most common observed mutations, respectively, in NRTI-, NNRTI-and PIassociated drug resistance. 93.76% subjects who had DRMs received the ART first-line regimens. CD4 count, symptoms in the past 3 months, and ART adherence were found to be associated with HIV-1 DR. Conclusions: This study showed that although the prevalence of HIV-acquired resistance in Hunan Province is at a low-level, the long-term and continuous surveillance of HIV ADR in antiretroviral drugs (ARVs) patients is necessary.