Nucleotide sequences of a part of the envelope glycoprotein B (gB) gene of human cytomegalovirus (CMV), encoding epitopes recognized by virus-neutralizing monoclonal antibodies, were determined for 12 distinct clinical strains of CMV after amplification of suitable templates using the polymerase chain reaction. Sequence analysis of this region (codons 384-717) revealed that the clinical strains and previously sequenced laboratory strains Towne and AD169 belong to one of four variant groups, each with a characteristic nucleotide and peptide sequence. Peptide homology was greater than 99% for strains within a group, and varied from 91% to 98% for strains in different groups. Variation was most frequent between codons 448 and 480. The gB group of a CMV strain could be determined by restriction analysis of a small target sequence amplified from viral genomic DNA, and an additional 28 clinical strains were grouped in this manner. The existence of a limited number of variants of gB among clinical strains facilitates analysis of biologic function and cross-reactivity of immune responses.