Five very low birth weight infants developed systemic Candida albicans infection during a 6-week period in an urban neonatal intensive care unit. In an effort to assess whether a common source outbreak was present, the genomic DNA of the clinical isolates was compared by EcoRI and XbaI restriction fragment analysis and Southern blot hybridization with 27A, a species-specific, transposon-like probe. Although the restriction fragment analysis suggested strong similarities among the isolates, the hybridization patterns demonstrated that all strains were genotypically distinct. The parallel use of at least two restriction enzymes was important for differentiating the isolates. Rapid genotypic analysis of clinical isolates from a cluster of C. albicans infections permits determination of whether a common source is probable, facilitates epidemiologic decisions and may reduce infection control measures and attendant costs.