Antiviral susceptibilities to ganciclovir, foscarnet, and cidofovir and sequencing of UL97 and DNA polymerase were done on 23 cytomegalovirus (CMV) isolates from 10 immunocompromised persons with end-organ CMV disease who were treated with ganciclovir alone or ganciclovir followed by foscarnet. Screening of UL97 for ganciclovir resistance mutations was done by restriction digest analysis. Of 14 isolates resistant to ganciclovir, 11 (79%) contained one or more UL97 mutations at codons known to confer resistance to this compound, and 10 (91%) had a concordant mutant pattern by restriction digest analysis. Of 9 isolates containing mutations in conserved regions of the DNA polymerase, 8 were resistant to ganciclovir, and 4 were cross-resistant to cidofovir. All isolates were susceptible to foscarnet. It is concluded that ganciclovir-resistant clinical CMV isolates may contain UL97 mutations, DNA polymerase mutations, or mutations in both genes. Ganciclovir therapy may select for CMV isolates that are cross-resistant to cidofovir.