Arbitrarily primed polymerase chain reaction (AP-PCR) was used to genotype Clostridium difficile isolates from various sources. Four major molecular types were identified among strains from the American Type Culture Collection previously typed by serogroup and from isolates from patients at the University of California, Davis Medical Center, from a patient at a Utah institution, and from the environment. These groups contained subgroups that displayed, in addition to the common group bands, at least one unique band. Two strains isolated from patients at our institution had the same DNA banding patterns. These patients were hospitalized during the same period, raising the possibility of cross-infection through hospital contact or another common source. These results suggest that this AP-PCR approach will be useful in epidemiologic studies of C. difficile infections.