To analyze the hepatitis B virus (HBV) quasi-subgenotype B3 characters and molecular evolution in Southeast Asia, 411 serum samples with HBsAg positive were collected from Xishuangbanna, China. After DNA extraction, PCR amplification and sequencing, a total of 183 HBV full-length genomes were obtained. Phylogenetic analysis showed 139 stains (76.0%) were genotype B, 41 strains were genotype C (22.4%) and 3 strains were genotype I (1.6%). Among genotype B, 34 sequences were identified as quasi-subgenotype B3. Quasi-subgenotype B3 sequences from this study and quasi-subgenotype B3 sequences from the GenBank (total of 141 complete genome sequences) were grouped into quasi-subgenotype B3 (B3, formerly B5, Chinese B6 and B7-9). Sixteen peculiar nucleotides distributed in quasi-subgenotype B3 were identified, which were differ from B1, B2, B4 and B5(formerly B6) (nt93 T, nt100C, nt355 G, nt843 T, nt861C, nt912C, nt929 T, nt930 G, nt1023 T, nt1041 T, nt2651C, nt2693 T, nt2970C, nt3054A, nt3087A and nt3171 G). Then Evolutionary dynamics analysis of HBV quasi-subgenotype B3 was performed. The mean rate of nucleotide substitution for HBV quasi-subgenotype B3 was estimated to be around 5.556-5.660 x 10(-4) substitutions/site/year. Estimated time to most recent ancestor of quasi-subgenotype B3 was around the 1847-1945(95%HPD), and Yunnan strains might be the parental strains. The Bayesian sky plot showed a steady spreading of HBV quasi-genotype B3 from early of 1940s to 90 s. In summary, HBV quasi-subgenotype B3 infection is prevalent in Southeast Asia based on the current reports and still with a high prevalence rate based on the evolutionary dynamics analysis. Current vaccine and nucleotide analogues might have effective prevention and treatment for HBV quasi-subgenotype B3 based on the rare clinically relevant mutation sites included in quasi-subgenotype B3.