Background Drug resistance has become a major concern for antiviral therapy in patients with chronic hepatitis B (CHB), leading to an increased risk of treatment failure. We aimed to identify the prevalence of hepatitis B virus (HBV) DNA polymerase mutations and to determine the frequency of HBV genotypes and subgenotypes in treatment-naive patients with CHB in Mekong Delta, Vietnam. Methods Of the 550 drug-naive CHB patients from Mekong Delta, their serum samples were analyzed for HBV DNA mutations, genotypes, and subgenotypes using polymerase chain reaction sequencing. Results Of the 550 patients who were infected with either HBV genotype B (72.9%) or genotype C (27.1%), the frequencies of subgenotypes were 19% B1, 3.6% B2, 10.3% B3, 39.8% B4, 0.4% C1, and 26.9% C2. The only mutation that was detected in 54 patients (10.7%) was rtV207M. The rtV207M mutation was found among 13.9% (51/368) of genotype B and among 2.2% (3/137) of genotype C ( P < 0.0001). Discussion Genotype B and subgenotype B4 are predominant in Mekong Delta. Only 1 mutation rtV207M is discovered in our study. These findings suggest that it is unnecessary to test for HBV resistance mutation before delivering antiviral therapy.