Human cytomegalovirus (HCMV) is a ubiquitous pathogen that infects a variety of cell types in vivo. A region (referred to as UL/b') present in the Toledo strain of HCMV and low passage clinical isolates contains 22 additional genes, which are absent in the highly passaged laboratory strain AD169. One of these genes, UL146, encodes an α‐chemokine. PCR amplification and sequencing of this gene from serial samples obtained from transplant recipients and samples from infants with suspected congenital HCMV infection, revealed that UL146 is a hypervariable gene in vivo. However, genetic changes were highly conserved in individuals and in renal transplant recipients multiple genotypes of UL146 were present. The majority of strains characterized maintained the conserved ELRCXC motif present in the Toledo strain of HCMV. These results provide further evidence that AD169 does not represent the authentic virus in vivo and although Towne and Toledo are more representative, major genetic differences still exist. Mixed populations of HCMV strains occur in vivo so cloning of these strains is essential if an authentic genotype is to be defined. J. Med. Virol. 74:573–579, 2004. © 2004 Wiley‐Liss, Inc.