We sought to characterize the molecular epidemiology of gram-negative bacilli (GNB) causing infections in infants and associated with carriage on nurses' hands after hand hygiene was performed. From March 2001 to January 2003, GNB caused 192 (34%) of 562 hospital-acquired infections in the 2 participating neonatal intensive care units (NICUs) and were isolated from the hands of 45 (38%) of 119 nurses. Five species—Klebsiella pneumoniae, Escherichia coli, Pseudomonas aeruginosa, Serratia marcescens and Enterobacter cloacae, all of which were typed by pulsed-field gel electrophoresis—caused 169 (88%) of 192 of GNB infections. Overall, 58% of infections were caused by unique strains not cultured from other infants or nurses, and 31% of infections were part of unrecognized molecular clusters. In contrast, only 9% of strains that caused infections were cultured from nurses' hands. These data suggest that practices in addition to hand hygiene are needed to prevent horizontal transmission of GNB in the NICU.