Infection with human cytomegalovirus (CMV) is common and may have grave consequences in transplant recipients and congenitally infected children. Diagnosis of CMV infection is based on detection of specific antibodies and molecular assays. The incorporation of CMV serological assays into diagnostic algorithms requires careful evaluation and interpretation. Very few serological assays measure CMV infection by a specific strain. We developed an enzyme-linked immunosorbent assay (ELISA) using CMV-encoded UL144 as antigen. UL144 encodes for three major genotypes, A, B and C, and recombinants. The ELISA was developed with the three UL144 proteins and optimized as a multiplex assay. Sera from 55 positive and 59 negative CMV IgG, determined by the clinical microbiology laboratory, were used for evaluation and optimization. A cutoff optical density (OD) that distinguishes UL144 antibody-positive from -negative sera was established. Sera detected UL144 A, B, C and combination of these antigens. Assay threshold of 0.1 was established, and from a total of 303 sera, the overall sensitivity, specificity, positive and negative predictive values of the multiplex ELISA were 86.72% (95% CI 79.59% to 92.07%), 96.57% (92.69% to 98.73%), 94.40% (88.45% to 97.38%), and 91.60% (87.50% to 94.44%). The inter- and intraassay median coefficients of variation were 0.06 (IQR 0.56, 0.2) and 0.171 (IQR 0.038, 0.302). No cross reactivity was observed with HSV-positive CMV-negative sera. This ELISA gives simple and reproducible results for detection of anti-CMV UL144 IgG. It may assist in differentiating natural infection from CMV vaccines that lack UL144, and may provide an important tool for epidemiological studies of CMV strains.