Understanding the prevalence and evolution of HIV-1 drug resistance (DR) and associated mutation patterns is critical to implementing free antiretroviral therapy in Yunnan, the first antiretroviral treatment location in China. Here We provide a basis for understanding the occurrence and development of HIV-1 resistance in Yunnan and a theoretical foundational for strategy to delay HIV-1 drug resistance and achieve successful individualized treatment.Plasma samples from different cities/prefectures were collected at Yunnan Provincial Hospital of Infectious Disease from January 2010 to September 2016, and those from drug-resistant individuals were genotyped using in-house assays, 88 patients were selected for the study who had been on treatment for ≥6 months (and for whom drug resistance was then measured), and each patient had at least 3 genotype resistance tests and who were enrolled to analyze mutation and evolution of HIV resistance.264 Pol sequences of 88 patients were obtained. Drug resistance levels to eight drugs increased to varying degrees with prolonged treatment. Resistance to efavirenz (EFV) and etravirine (ETR) showed the highest change, comparisons of resistant changes to second and first and to third and second agents showed altered level of drug resistance were 25 and 20 cases, 28 and 18 cases, respectively. The smallest change was Lopinavir/Ritonavir (LPV/r) present 2 and 3 cases; Resistance to lamivudine (3TC) and lopinavir/ritonavir (LPV/r) was high among patients detected thrice, whereas other drugs were distributed in all resistance levels. M184 V/I (26.14%), T69S (11.36%), and T215Y/I (10.23%) mutations were the most common in nucleoside reverse transcriptase inhibitors (NRTIs), and K103 N/R/S (21.59%), V179D/E (20.45%) in Non-NRTIs (NNRTIs). Furthermore, L10 V/F/I (6.82%), A71V (4.55%), and I54V (4.55%) mutations were common in protease inhibitors (PIs).We found dynamic genotypic changes in HIV-1 drug-resistance in Yunnan, with prolonged treatment, and drug resistance was inevitable. However, resistance to different drugs occurred at varying times, and mutation site emergence was the main cause. These findings enhance our understanding of evolution and regulation, and are valuable for developing HIV-1 DR prevention strategies in Yunnan.