HIV drug resistance compromises the ability of anti-retroviral therapy (ART) to suppress viral replication, resulting in treatment failure. This study investigates the prevalence of pre-treatment drug resistance (PDR) in newly diagnosed individuals in a prosperous city (Wenzhou) in Southeastern China. A cross-sectional investigation was carried out among 473 newly diagnosed ART-naive HIV-1-infected individuals between January and December 2022. The protease–reverse transcriptase (PR-RT) region and integrase (IN) region of HIV-1 were amplified by two separately nested PCRs, followed by sequencing. Drug resistance mutations (DRMs) and drug resistance to nucleoside reverse transcriptase inhibitors (NRTIs), non-nucleoside reverse transcriptase inhibitors (NNRTIs), protease inhibitors (PIs) and integrase strand transfer inhibitors (INSTIs) were analyzed. The PDR prevalence was 6.5% [95% CI: 4.4–9.1] for any anti-retroviral drug, 0.9% [95% CI: 0.3–2.3] for NRTIs, 4.1% [95% CI: 2.5–6.5] for NNRTIs, 1.8% [95% CI: 0.8–3.6] for PIs and 0.5% [95% CI: 0.1–1.8] for INSTIs. According to the subtyping results of the PR-RT region, 11 different subtypes and 31 unique recombinant forms (URFs) were found. CRF07_BC was the dominant subtype (53.7%, 233/434), followed by CRF01_AE (25.3%, 110/434). V179D (1.6%) and K103N (1.4%) were the most predominant types of NNRTI DRMs. Q58E (1.2%) and M184V (0.7%) were the most frequent PI DRMs and NRTI DRMs, respectively. The INSTI-related DRMs Y143S (causes high-level resistance to RAL) and G163K (causes low-level resistance to EVG and RAL) were found in one patient each. Given the relatively high PDR prevalence of NNRTI (4.1%), non-NNRTI-based ART may be preferred in the future. It is recommended to include genotypic resistance testing before starting ART in regions where feasible.