The presence of pretreatment drug resistance (PDR) may jeopardize the success of standardized HIV management protocols in resource-limited settings. We evaluated the prevalence of and factors influencing PDR in Guangzhou, China, as well as the distribution of drug resistance mutations (DRMs) among HIV subtypes. Blood samples were collected from patients who initiated antiretroviral therapy (ART) from 2018 to 2022 in Guangzhou, China. PDR was analysed using HIV pol sequences. The prevalence of PDR in Guangzhou was 7.4%, with resistance rates to nucleotide reverse transcriptase inhibitor (NRTI), nonnucleoside reverse transcriptase inhibitor (NNRTI), and protease inhibitor (PI) of 1.3%, 4.8%, and 1.4%, respectively. Abacavir (0.8%) resistance was the most common in NRTI, followed by resistance to emtricitabine (0.6%), lamivudine (0.6%), and tenofovir disoproxil fumarate (0.3%). In NNRTI, nevirapine (3.7%) resistance was the most common, followed by efavirenz (3.5%) and rilpivirine (3.4%). In the multivariable logistic regression model, hepatitis B surface antigen positivity (adjusted odds ratio [aOR], 1.42; P<0.05), CRF55_01B (aOR, 3.39; P<0.001), CRF08_BC (aOR, 2.69; P<0.001), CRF59_01B (aOR, 1.90; P<0.05), and subtype B (aOR, 2.43; P<0.001) were demonstrated as influencing factors for PDR. CRF55_01B had the highest prevalence of DRMs (43.6%), followed by CRF01_AE (24.0%) and CRF07_BC (19.3%), whereas CRF07_BC (29.0%) showed the highest rate of drug resistance. The overall prevalence of PDR in Guangzhou was moderate, with relatively severe NNRTI resistance. HIV subtype is a significant risk factor for PDR. Therefore, monitoring of PDR and HIV subtype should be strengthened.