Objective To compare the application of nucleotide drugs and the virology characteristics between patients with only N236T mutation and patients with N236T+A181T mutation in the HBV reverse transcriptase(rt) region. Methods Sera of patients with only rtN236T mutation and patients with rtN236T+rtA181T mutation were obtained from patients with chronic hepatitis B. Then the levels of sero-HBsAg, HBV DNA and ALT were determined and the HBV genotypes were analyzed. The treatment history with nucleotide drugs was retrospectively reviewed. Results The sero-HBsAg levels were not significantly different between only rtN236T mutation group and rtN236T+rtA181T mutation group (P=0.975 5), and significantly higher viral replication (P=0.001 4) and higher ALT level (P=0.003 2) were found in rtN236T+rtA181T mutation group. Moreover, compared to HBV B genotype, patients with HBV C genotype were prone to carry rtN236T+rtA181T mutation(40% vs 20.45%, P=0.023 5); also we noticed that a switch from lamivudine medication to adefovir medication was liable to induce the virus mutation. Conclusion Nucleotide drug application in HBV patients with only rtN236T mutation and rtN236T+rtA181T mutation are concurrent (lamivudine switching to adefovir). However, the HBV genotype constituents and the serum virological characteristics are different between the two types of HBV patients.