Neonatal necrotizing enterocolitis (NEC) is an inflammatory intestinal disorder affecting preterm infants. Intestinal bacteria have an important function; however no causative pathogen has been identified. The purpose of this study was to determine if there are differences in microbial patterns that may be critical to the development of this disease. Fecal samples from 20 preterm infants, 10 with NEC and 10 matched controls (including 4 twin pairs) were obtained from patients in a single site level III neonatal intensive care unit. Bacterial DNA from individual fecal samples was PCR-amplified and subjected to terminal restriction fragment length polymorphism analysis and library sequencing of the 16S rRNA gene to characterize diversity and structure of the enteric microbiota. The distribution of samples from NEC patients distinctly clustered separately from controls. Intestinal bacterial colonization in all preterm infants was notable for low diversity. Patients with NEC had even less diversity, an increase in abundance of Gammaproteobacteria, a decrease in other bacteria species, and had received a higher mean number of previous days of antibiotics. Our results suggest that NEC is associated with severe lack of microbiota diversity that may accentuate the impact of single dominant microorganisms favored by empiric and widespread use of antibiotics.