Objective to investigate the association between HBV genotypes and characteristics of rtA181 mutation. Methods Total of 85 chronic hepatitis B (CHB) patients who appeared rtA181 mutation after nucleos(t)ide analogs (NAs) therapy were enrolled in this study. Levels of serum ALT, AST, HBV DNA and HBsAg titers were monitored during therapy. HBV reverse transcriptase genes were amplified and sequenced to identify genotypes and resistance mutations. Virions and HBsAg in HepG2 cell with rtA181 mutation were also compared between genotypes B and C. Results The majority of sera contained HBV genotypes B (15.7%) and C (84.3%). There were no significant difference of rtA181 mutant patterns between genotypes (P > 0.05). After emergence of rtA181 mutation, serum ALT, AST, HBV DNA levels and HBsAg titers were decreased than that at baseline (P < 0.05), while these characteristics were not different between genotypes B and C (P > 0.05). In cellular experiment, there were no significant differences between genotypes B and C not only in HBV virions but also in HBsAg titres (P > 0.05). Conclusions No differences of clinical characteristics and cellular results were found in rtA181 mutation of HBV genotypes B and C.