The purpose of this study was to determine the prevalence of ganciclovir (GCV) resistance-conferring human cytomegalovirus (HCMV) UL97 gene mutations and UL97 polymorphisms in Korean immunocompromised patients. A partial sequence of the HCMV UL97 gene spanning codons 430 to 644 was amplified in 77 samples from 32 patients by nested polymerase chain reaction (PCR) and sequenced directly. A cysteine-to-glycine mutation at codon 592 (C592G) conferring GCV resistance was detected in a 2-year-old girl after a 40-day GCV treatment, but overall, UL97 gene mutations associated with GCV resistance were rare in GCV-treated patients. An aspartate-to-glutamate substitution at codon 605 (D605E) was detected in 29 of 32 patients (90.6%), but 17 of 19 (89.5%) GCV-naïve patients also possessed D605E. These findings indicate that the D605E polymorphism, which is frequent in Korean patients and thus may be a natural sequence variant, could be a genetic marker for HCMV in Asian countries.