Human immunodeficiency virus (HIV)/hepatitis B virus (HBV) co-infection accelerates the progression of HBV-related liver diseases. HBV basic core promoter (BCP)/pre-core (preC) gene mutations may be one of the most important risk factors. In this study, a total of 230 patients were recruited, and 199 patients whose HBV BCP/preC gene were successfully amplified and sequenced, including 99 HIV/HBV co-infected and 100 HBV mono-infected patients. Next-generation sequencing was used for detection of BCP/preC mutations which were then compared in patients with different HBV genotypes and different HBeAg statuses, and 1% and 20% cutoff values were defined to evaluate the mutations. HBV quasispecies diversity was also compared in HIV/HBV co-infected and HBV mono-infected patients. Among the patients infected with HBV genotype C and HBeAg-negative status, the frequency of A1762T/G1764A double mutations was significantly lower in HIV/HBV co-infected patients than in HBV mono-infected patients (53.3% vs. 100.0%, P = 0.008) regardless of the 1% or 20% cutoff value level. However, A1762T/G1764A double mutations did not differ in the other groups (P >0.05). Viral quasispecies diversity was lower in HIV/HBV co-infected patients than in HBV mono-infected patients (P Keywords: human immunodeficiency virus, hepatitis B virus; mutations; viral quasispecies; next-generation sequencing.