The role of atypical enteropathogenic Escherichia coli (EPEC) in childhood diarrhea is controversial. The aim of the present study was to search for genes linked with diarrhea in atypical EPEC strains from a case-control study among Norwegian children. Using DNA microarray analysis, genomic DNAs from strains isolated from children with (n = 37) and without (n = 20) diarrhea were hybridized against 242 different oligonucleotide probes specific for 182 virulence genes or markers from all known E. coli pathotypes. PCR was performed to test the strains for seven putative virulence genes not included in the microarray panel. The OI-122 gene efa1/lifA was the gene with the strongest statistical association with diarrhea (P = 0.0008). Other OI-122 genes (set/ent, nleB, and nleE) and genes with other locations (lpfA, paa, ehxA, and ureD) were also associated with diarrheal disease. The phylogenetic marker gene yjaA was negatively associated with diarrhea (P = 0.0004). Atypical EPEC strains could be classified in two main virulence groups based on their content of OI-122, lpfA, and yjaA genes. Among children with diarrhea, atypical EPEC isolates belonging to virulence group I (OI-122 and lpfA positive, yjaA negative) were the most common, while the majority of isolates from healthy children were classified as virulence group II strains (OI-122 negative, lpfA and yjaA positive; P < 0.001). In conclusion, using DNA microarray analysis to determine the virulence gene profile of atypical EPEC isolates, several genes were found to be significantly associated with diarrhea. Based on their composition of virulence genes, the majority of strains could be classified in two virulence groups, of which one was seen mainly in children with diarrhea.