Human cytomegalovirus (HCMV) isolates resistant to ganciclovir were found in patients undergoing therapy. Therefore, we have developed a new specific and sensitive method--a ligase chain reaction (LCR) assay--for detection of frequently encountered 594 mutated codon in ganciclovir (GCV) resistant virus. Previous studies characterized an alanine to valine change on codon 594 in resistant strains. A novel substitution in 594, alanine to glycine, is described which is also capable of conferring ganciclovir resistance. LCR products were analyzed on polyacrylamide gel- and the mutant was detected using a non radioactive method. The LCR product detection was then adapted to a microtitre plate format with a colorimetric detection. This method allowed the distinction of mutated GCV-resistant strains from sensitive strains with a high sensitivity, and the detection of a low percentage of mutated DNA in virus load. This assay could be useful in following the evolution of mutated DNA compared to viral infection.