Intestinal colonization withKlebsiellahas been linked to necrotizing enterocolitis (NEC), but methods of analysis usually failed to discriminateKlebsiellaspecies or strains. A novel ~ 2500-base amplicon (StrainID) that spans the 16S and 23S rRNA genes was used to generate amplicon sequence variant (ASV) fingerprints forKlebsiella oxytocaandKlebsiella pneumoniaespecies complexes (KoSC and KpSC, respectively) and co-occurring fecal bacterial strains from 10 preterm infants with NEC and 20 matched controls. Complementary approaches were used to identify cytotoxin-producing isolates of KoSC.Klebsiellaspecies colonized most preterm infants, were more prevalent in NEC subjects versus controls, and replacedEscherichiain NEC subjects. Single KoSC or KpSC ASV fingerprinted strains dominated the gut microbiota, suggesting exclusionaryKlebsiellacompetition for luminal resources.Enterococcus faecaliswas co-dominant with KoSC but present infrequently with KpSC. Cytotoxin-producing KoSC members were identified in most NEC subjects and were less frequent in controls. FewKlebsiellastrains were shared between subjects. We conclude that inter-speciesKlebsiellacompetition, within an environment of KoSC andE. faecaliscooperation, appears to be an important factor for the development of NEC. Preterm infants seem to acquireKlebsiellaprimarily through routes other than patient-to-patient transmission.