OBJECTIVE: To characterize the epidemiological relationships among Stenotrophomonas maltophilia isolates in the neonatology unit of our institution over a 4-month period in which an increased number of isolates was observed. SETTING: The neonatology ward in a 2,000-bed university hospital in Madrid, Spain. DESIGN: A retrospective molecular epidemiological analysis using three different typing methods, arbitrarily primed polymerase chain reaction (PCR), pulsed-field gel electrophoresis, and enterobacterial repetitive intergenic consensus-PCR, was performed with 11 isolates obtained from seven neonates over a 4-month period. Presumed unrelated isolates also were included as controls. A similarity dendrogram was obtained, to analyze the genetic relatedness among the isolates. RESULTS: All isolates from the neonates, except one, showed a remarkably high homology among their typing patterns for the three methods assayed and clustered in the relatedness dendrogram at 96% similarity. The unrelated strains selected as controls were unclustered. The index case was considered to be a newborn who had an S maltophilia isolate from a culture drawn on the day of admission to the neonatology unit and which was included in the clustered similarity group. CONCLUSIONS: Such a high genetic similarity among the isolates, together with the presence of an index case who had been colonized or infected by S maltophilia before arrival at our institution, constitutes the first evidence of nosocomial cross-transmission of this microorganism (Infect Control Hosp Epidemiol 1999;20:816-820).