Chronic hepatitis B (CHB) is one of the most common infections worldwide. Currently approved treatments of CHB include nucleoside/nucleotide analogues (NAs). However, long-term NA therapy is associated with accumulation of resistant mutations within the hepatitis B virus (HBV) polymerase gene. The incidence of naturally occurring HBV mutations leading to primary antiviral resistance has not been fully elucidated yet. The objective of present study was to detect the frequency of mutations within the HBV polymerase gene in 263 patients naïve to nucleoside/nucleotide analogues. Prevalence of HBV Pol gene mutations secondary to NA treatment in patients without pre-existing antiviral resistance mutations was also examined. Retrospective analysis showed that HBV Pol gene mutations were present in 7 out of 263 patients prior to the treatment. Mutations observed in NA-naïve CHB patients were associated only with resistance to lamivudine and adefovir. Compensatory mutations were observed as well. In the course of antiviral treatment, HBV Pol gene mutations were identified in 65 out of the remaining 256 CHB patients (25.39%), while no mutations of any type were detected in 160 patients (62.5%). The profiles of detected mutations were comparable to those observed in other studies that focused on the analysis of clinically relevant NA-resistant mutations. In conclusion, we found out that antiviral resistance mutations may pre-exist in the overall viral population present in untreated patients, although the incidence of HBV Pol gene mutations in NA-naïve CHB patients was low and reached only up to 2.66%. However, possible circulation and transmission of NAs-resistant HBV mutants in human population should be taken into account.