Molecular typing techniques have been used in outbreak investigations. In this study, molecular typing techniques were used to track the spread of gram-negative rods (GNRs) in a neonatal intensive care unit (NICU) in the absence of an outbreak. Stool or rectal swab cultures for GNRs were obtained from all infants on admission, weekly, and on discharge. GNRs were tested for gentamicin susceptibility and were typed by contour-clamped homogeneous electric field electrophoresis. Transmission of identical strains of GNRs among infants was noted. Shared strains were more gentamicin resistant compared with unique strains (53% vs. 10%; P=.0001). Infants first colonized when they were >1 week of age had more total days of antibiotic treatment and had a higher rate of acquiring a shared and gentamicin-resistant strain, compared with infants colonized earlier. Antibiotic use increases colonization of infants in the NICU with resistant and shared strains of GNRs.