AbstractClostridium perfringensis an anaerobic toxin-producing bacterium associated with intestinal diseases, particularly in neonatal humans and animals. Infant gut microbiome studies have recently indicated a link betweenC. perfringensand the preterm infant disease necrotizing enterocolitis (NEC), with specific NEC cases associated with overabundantC. perfringenstermedC. perfringens-associated NEC (CPA-NEC). In the present study, we carried out whole-genome sequencing of 272C. perfringensisolates from 70 infants across 5 hospitals in the United Kingdom. In this retrospective analysis, we performed in-depth genomic analyses (virulence profiling, strain tracking and plasmid analysis) and experimentally characterized pathogenic traits of 31 strains, including 4 from CPA-NEC patients. We found that the gene encoding toxin perfringolysin O,pfoA, was largely deficient in a human-derived hypovirulent lineage, as well as certain colonization factors, in contrast to typicalpfoA-encoding virulent lineages. We determined that infant-associatedpfoA⁺strains caused significantly more cellular damage thanpfoA⁻strains in vitro, and further confirmed this virulence trait in vivo using an oral-challenge C57BL/6 murine model. These findings suggest both the importance ofpfoA⁺C. perfringensas a gut pathogen in preterm infants and areas for further investigation, including potential intervention and therapeutic strategies.