Objective To investigate the difference in naturally occurring resistance-associated variants (RAVs) between the patients with HIV/HCV co-infection and those with HCV infection alone by detecting the drug resistance loci associated with HCV NS3/4A protease and NS5A inhibitors. Methods A total of 246 patients with HIV/HCV co-infection or HCV infection alone who were hospitalized or attended the outpatient service in Guangzhou Eighth People's Hospital, Guangzhou Medical University, from January 2016 to January 2020 were enrolled in this study. Serum samples were collected and next-generation sequencing (Illumina platform, PE250) was used for sequencing. The two groups of patients were compared in terms of RAVs associated with NS3/4A protease and NS5A inhibitors approved in China, and the drugs for analysis included asunaprevir/daclatasvir (ASV/DCV) and elbasvir/grazoprevir (EBR/GZR) for HCV genotype 1b and glecaprevir/pibrentasvir (GLE/PIB) for pan-genotypes. The t -test was used for comparison of normally distributed continuous data between two groups, and the Mann-Whitney U test was used for comparison of non-normally distributed continuous data between two groups; the chi-square test or the Fisher's exact test was used for comparison of categorical data between two groups. Results Among the 246 serum samples included in this study, 239 samples (97.2%) were successfully amplified by PCR and sequenced, with 102 samples from the patients with HIV/HCV co-infection and 137 from the patients with HCV infection alone. The analysis of RAVs associated with ASV/DCV and EBR/GZR showed that Y56F, Q80K/L, and S122N/R/T associated with ASV and GZR and L31M and Y93H associated with DCV and EBR were observed in patients with HIV/HCV (genotype 1b) co-infection or HCV (genotype 1b) infection alone; 2 patients with HIV/HCV co-infection had the RAVs of Y56F+Y93H associated with EBR/GZR, and 2 with HCV infection alone had the RAVs of Q80L+L31M and Y56F+Y93H, respectively, associated with EBR/GZR, with no significant difference in RAVs between the two groups (both P > 0.05). The analysis of RAVs associated with GLE/PIB for pan-genotypes showed that 3 patients with PIB-associated Y93H RAV were observed among the patients with HCV genotype 3a infection, among whom 2 had HIV/HCV co-infection and 1 had HCV infection alone ( P =0.590), and in addition, no RAVs associated with GLE/PIB were observed. Conclusion There is no significant difference in naturally occurring RAVs associated with HCV NS3/4A protease and NS5A inhibitors between the patients with HIV/HCV co-infection and those with HCV infection alone.