To gain insight into hepatitis B virus (HBV) transmission in the Netherlands, epidemiological data and sera were collected from reported cases of acute HBV infections in the Netherlands in 2004. Cases were classified according to mode of transmission. A fragment of the S-gene of HBV (648 bp) was amplified, sequenced, and subjected to phylogenetic analysis. Of the 291 acute HBV cases reported in 2004, 158 (54%) were available for genotyping. Phylogenetic analysis identified 6 genotypes: A (64%), B (3%), C (3%), D (21%), E (5%) and F (5%). Of HBV infected men having sex with men, 86% were infected with genotype A, accounting for 43% of all patients infected with this genotype. There were only three reported cases of injecting drug use of which one was available for sequencing (genotype A). Unlike the genotype A cluster, sequences within the genotype B-E clusters were heterogenic. Within genotype F, several isolates had identical sequences, but patients could not be epidemiologically linked. Sexual transmission, particularly by men having sex with men was the most important transmission route for HBV. Injecting drug use plays a minor role. Genotype A is predominant in the Netherlands, especially among men having sex with men. In addition to imported strains, there seems to be a pool of related but non-identical strains circulating among chronic carriers in the migrant population, from which occasionally new patients are infected, primarily by heterosexual transmission.