Virological data on chronic hepatitis B virus (HBV) infection in Greece are limited. HBV genotypes, surface antigen (HBsAg) subtypes, and HBsAg "a" determinant mutations among patients infected chronically with HBV, were investigated. Serum samples from 135 HBsAg positive patients were tested. Serologic (HBsAg, anti-HBs, HBeAg, and anti-HBe), virologic (HBV-DNA quantitation) and biochemical markers (serum alanine aminotransferase/ALT and aspartate aminotransferase/AST) were analyzed. HBV genotypes and HBsAg subtypes were determined by partial sequencing of the S gene. Genotyping was performed by using the National Center for Biotechnology Information online Genotyping tool and phylogenetic analysis. Nucleotide sequences were aligned pair wise with ClustalW and phylogenetic trees were constructed by the neighbor-joining method. Sequences were also used to predict HBV HBsAg subtypes. In six patients (4%), simultaneous presence of HBsAg and anti-HBs was determined, whereas 47 patients (35%) were HBeAg positive, 84 (62.5%) were anti-HBe positive, and four patients (3%) were characterized by the simultaneous presence of HBeAg and anti-HBe. Mean ALT was 238 IU/L (standard deviation = 576.84), and HBV-DNA levels ranged from 1.02 × 10(5) to 2.2 × 10(7) IU/ml. Genotype D was predominant (98%), with viral groups D/ayw2 (73%) and D/ayw3 (27%). Group A/adw accounted for 1% of cases. Genotypes B and C were found exclusively in the Chinese immigrants (1%). Single or multiple point mutations were found in 35 cases (26%). Some of the most common mutations occurred at amino acid positions 129, 133, 134, 144, 145, including the "vaccine escape" mutation G145R. Mutations analysis revealed that amino acid substitutions did not affect detection by commercial immunoassays.