Although the prevalence of pretreatment HIV drug resistance (PDR) in China is rising, the evidence of potential PDR on ainuovirine (ANV)-based antiretroviral treatment (ART) is inconclusive. This study aimed to evaluate the impact of potential PDR on the virological outcomes of ANV-based ART. A randomized, double-blind, double-dummy, positive parallel group, phase 3 trial was conducted in 7 sites across China. Plasma samples were collected and the entire pol and IN gene were amplified and sequenced using HIV genotypic resistance testing. Patients with successful sequencing were followed up for 48 weeks to observe the virological outcomes of ART. Between November 27, 2018 and March 11, 2021, a total of 630 were finally enrolled and randomly assigned (1:1) to either ANV (n = 315) or EFV (n = 315) groups. Among the 591 included study participants, PDR was detected in 3.9% (n=23). 20 (3.4%) were resistant to NNRTIs and 5 (0.84%) were resistant to NRTIs. The virological suppression (VS) rate of the no PDR group and the ANV group had potential PDR to NNRTIs were 95% and 96.6% respectively, which was significantly higher than that of the EFV group had potential PDR to NNRTIs (90%). ANV exhibits significantly better antiviral activity compared to EFV and the prevalence of potential PDR negatively impacts outcomes of ANV-based ART.