A technique based on polymerase chain reaction (PCR) amplification was developed to facilitate the study of the epidemiology of cytomegalovirus (CMV). Consensus oligonucleotide primers from repetitive DNA sequences were designed to amplify interspersed repetitive sequences in an area of heterogeneity within the L-S junction region of the CMV genome, and PCR products were detected by gel electrophoresis. Purified CMV DNAs from 25 CMV isolates, 13 from members of five families in which person-to-person transmission was documented, 9 random clinical isolates of CMV, and 3 laboratory reference strains of CMV (Towne, Davis, and AD169), were analyzed. The gel electrophoretic patterns of DNA bands, or PCR profiles, produced by amplification with the L-S primers were unique for epidemiologically unrelated strains and laboratory reference strains, yet similar patterns were observed for epidemiologically related strains isolated from members of the same family. This method of rapid fingerprinting of CMV DNA within the hypervariable L-S junction region by PCR to produce strain-specific, variably sized PCR products should simplify the molecular epidemiologic analysis of CMV.