Reliable methods for the detection of cytomegalovirus (CMV) strain-specific serological responses are lacking. We describe a simple and reliable enzyme-linked immunosorbent assay method developed to detect antibodies against the polymorphic epitopes within the two envelope glycoproteins of CMV, glycoproteins H and B. This assay is useful for the detection of serologic responses to CMV strains and the identification of CMV reinfections. © 2009, American Society for Microbiology. All Rights Reserved.