To evaluate the colonization of Candida species and the importance of cross-contamination with Candida albicans, we prospectively screened clinical specimens obtained from surgical patients in the intensive care unit (ICU) who had a high risk of yeast colonization. Genotyping of C. albicans was performed using microsatellite markers. Thirty-six of 94 patients acquired nosocomial yeast colonization and/or infection. A total of 1126 specimens were cultured, 167 (15%) of which yielded yeasts. All 122 isolates of C. albicans recovered from the 30 C. albicans-positive patients were genotyped. Twenty-four different genotypes were identified. No genotype was systematically associated with a specific room or time. Isolates recovered from different body sites of patients at different times had identical genotypes. Acquisition of C. albicans in the surgical ICU seems to be mainly endogenous. Microsatellite markers should also be developed for typing non-albicans Candida species to learn whether their epidemiology differs from that of C. albicans.