Human cytomegalovirus (HCMV) strains display genetic polymorphisms, and these polymorphisms can be analyzed to study viral transmission and pathogenesis. Recently, short tandem repeat (STR) length polymorphisms have been identified in the HCMV genome. We assessed the utility of STRs in characterizing HCMV strains and found that a multiplexed PCR assay using primers based upon these STRs accurately maps HCMV strains. Using primers for 10 microsatellite regions, the STR profiles of 44 wild-type and 2 laboratory strains of HCMV were characterized. The results of STR analysis were compared with those for strain characterization using nucleotide sequencing and restriction fragment length polymorphism analysis. In each instance, STR analysis accurately and specifically identified strains that were indistinguishable or distinct by conventional molecular analysis. Analysis of short tandem repeats also detected polymorphisms that supported simultaneous excretion of two HCMV strains. These results indicate that STR analysis allows rapid, precise molecular characterization of HCMV strains.Human cytomegalovirus (HCMV), a betaherpesvirus, has a large, complex genome consisting of approximately 240,000 nucleotide base pairs (bp) and over 200 open reading frames (20). Wild-type and laboratory strains of HCMV display nucleotide polymorphisms in several regions of the genome, especially in the a sequence, major immediate early (MIE), glycoprotein B (gB; UL55), and UL144 gene regions (7,8,12,(16)(17)(18); J. F. Bale, S. J. Petheram, M. Robertson, J. R. Murph, and G. Demmler, submitted for publication). Although the effects of these polymorphisms on the biology of HCMV infections are largely unknown, molecular variations can be analyzed to study the epidemiology and pathogenesis of HCMV infections (4,5,16).Mapping the molecular profiles of HCMV has enabled investigators to determine the source and transmission patterns of HCMV infections. Studies of HCMV epidemiology have mapped strains by using analysis of the restriction fragment length polymorphisms (RFLPs) of the entire HCMV genome (1, 10), PCR-based analysis of a sequence variations (2, 22), DNA sequence analysis of polymorphic regions (11,19; Bale et al., submitted), and stepwise analysis of the RFLPs of gene regions by using PCR-based methods (3,7,15,18). Our laboratory has previously used a PCR-based algorithm that compares the size and RFLPs of the a sequence amplicon, the gB genotype as described by Chou and Dennsion (8), and the RFLPs of an amplicon derived by amplification of the MIE gene region (18). Although these molecular approaches can characterize HCMV strains accurately, they are time-consuming and available only in research laboratories.Recently, Davis and colleagues reported that the HCMV genome contains numerous short tandem repeats (STRs) (9). The microsatellite regions consist of iterated motifs of one to six bases that occur in the genomes of eukaryotes and some prokaryotes and represent potential sites of mutation (14, 21). Davis and colleagues found at ...