The transmission of infectious diseases can be traced using epidemiological and molecular information. In the current study, the congruence was assessed between sequence data of the hepatitis B virus (HBV) and epidemiological information resulting from source and contact tracing of patients seen at the Municipal Public Health Service in Rotterdam between 2002 and 2005. HBV genotypes A-G were present in 62 acute and 334 chronic HBV patients. At the sequence level, the identical sequences of members of epidemiological transmission pairs and the rarity of such pairs provided strong support for correctness of the hypothesized transmission routes. The molecular support for epidemiological transmission pairs derived from source and contact tracing was further assessed by using topological constraints in parsimony analyses in agreement with epidemiological information, and by taking the presence of polymorphic sites of HBV within patients into account. This, in principle, allows mutations in epidemiological clusters. Of 22 epidemiological clusters, six could be refuted, four clusters received support from the molecular analysis, and support for the remaining twelve clusters was ambiguous. Two of the four epidemiological pairs that received molecular support had diverged (by 3 and 15 mutations). These results show that levels of divergence cannot be used simply as an indicator of the likelihood that groups of sequences constitute transmission pairs. Instead, to confirm or refute transmission pairs, it is necessary to assess the likelihood of a common origin of HBV variants in epidemiologically defined transmission groups relative to the HBV diversity in the local community.