Chronic hepatitis B (CHB) is treated with nucleos(t)ide analogs (NA). The reverse transcriptase (RT) region in the Hepatitis B virus (HBV) genome mutates to resist NA treatment, yet the RT mutations have not been well characterized. Further, the HBV genotype might influence RT sequence evolution, NA resistance (NAr) mutation patterns, and drug resistance development. We examined 42 NAr mutation sites in 169 untreated and 131 NA-treated CHB patient samples. Patients were identified with genotype B (HBV-B) and HBV-C infections, with a higher prevalence and mutation frequency of HBV-C than HBV-B. Seventeen reported NAr mutation sites and 13 novel mutations were detected. NAr-related mutation prevalence was significantly higher in NA-treated versus untreated patients. Primary antiviral-resistant mutants only existed in NA-treated patients. Sequencing data revealed 7 HBV-C-specific mutations and 3 HBV-B-specific mutations. In conclusion, NA treatment and HBV genotype might constitute the selection basis and promote NA-resistant HBV strain evolution under antiviral therapy.