Necrotizing enterocolitis (NEC) is one of the most common gastrointestinal emergencies in premature infants and has been linked with viral antigens in as much as 40% of cases in single-center cohorts. We examined 28 tissue sections from surgically resected ileum from 27 preterm infants with NEC from 2 separate institutions for 15 common bacterial, viral, and parasitic gastrointestinal pathogens using multiplex reverse transcriptase polymerase chain reaction amplification and suspension array detection methods. We did not detect infectious enteritis pathogens in any of the NEC tissues and conclude that gastrointestinal pathogens are a rare cause of NEC.