An epidemiologically linked cluster of hepatitis B virus (HBV) infections was investigated using HBV DNA amplification by a nested polymerase chain reaction with primers complementary to the region around the immunodominant a determinant of the surface gene, part of the X and core genes, and precore region and direct nucleotide sequence analysis. The cluster, in which 2 persons died of fulminant hepatitis, comprised 1 blood donor, 2 patients, and 2 health care workers. The Kimura two-parameter method was used to compare variance among the cluster with that in the control samples, which were collected from 7 patients infected with the same HBV subtype. Significantly less variation occurred within the cluster than in the control group (unpaired t test, P < .05). In an unrooted phylogenetic tree analysis, the 5 study samples formed a cluster distinct from the controls. This direct molecular approach of analyzing conserved regions of the HBV genome differentiated between viruses involved in HBV transmission events.