Objective: To investigate HIV-1 subtypes and drug resistance in HIV/AIDS patients who failed in antiretroviral therapy in Jinhua City, Zhejiang Province, so as to provide the basis for improving antiretroviral therapy strategy. Methods: Totally 128 plasma samples of HIV/AIDS patients who failed in antiretroviral therapy (treatment for more than 6 months and viral load ≥1 000 copies/mL) from January 1 to November 30, 2023 were collected. After nucleic acid extraction and amplification, the sequences of HIV-1 pol genes were determined using first generation sequencing method, then submitted to HIV resistance database of Stanford University in the United States, and the subtypes, drug resistance mutations and drug resistance status of HIV-1 were analyzed. Results: A total of 118 sequences of HIV/AIDS patients were obtained, including 94 males and 24 females (male to female ratio, 3.9︰1). There were 53 cases aged between 40 to 59 years, accounting for 44.92%. The main infection routes was heterosexual contact, with 92 cases accounting for 77.97%. The main HIV-1 gene subtypes were CRF07_BC and CRF01_AE, with 45 and 39 cases accounting for 38.14% and 33.05%, respectively. There were 75 cases found to have drug-resistant site mutations, with a mutation rate of 63.56%. The most common mutation sites were M184 and K103, with mutation rates of 29.66% and 28.81%, respectively. There were 58 cases with resistance to more than one drug, with a rate of 49.15%. The rates of resistance to non-nucleoside reverse transcriptase inhibitors (NNRTI), nucleotide reverse transcriptase inhibitors (NRTI) and protease inhibitors (PI) were 50.00%, 33.90% and 4.24%, respectively. Conclusion The HIV-1 gene subtypes of HIV/AIDS patients who failed in antiretroviral therapy in Jinhua City are mainly CRF07_BC and CRF01_AE, which are mainly resistant to NNRTI and NRTI.