BACKGROUND & AIMS: The intestinal microbiomes of healthy children and pediatric patients with irritable bowel syndrome (IBS) are not well defined. Studies in adults have indicated that the gastrointestinal microbiota could be involved in IBS. METHODS: We analyzed 71 samples from 22 children with IBS (pediatric Rome III criteria) and 22 healthy children, ages 7-12 years, by 16S ribosomal RNA gene sequencing, with an average of 54,287 reads/stool sample (average 454 read length = 503 bases). Data were analyzed using phylogenetic-based clustering (Unifrac), or an operational taxonomic unit (OTU) approach using a supervised machine learning tool (randomForest). Most samples were also hybridized to a microarray that can detect 8741 bacterial taxa (16S rRNA PhyloChip). RESULTS: Microbiomes associated with pediatric IBS were characterized by a significantly greater percentage of the class γ-proteobacteria (0.07% vs 0.89% of total bacteria, respectively; P < .05); 1 prominent component of this group was Haemophilus parainfluenzae. Differences highlighted by 454 sequencing were confirmed by high-resolution PhyloChip analysis. Using supervised learning techniques, we were able to classify different subtypes of IBS with a success rate of 98.5%, using limited sets of discriminant bacterial species. A novel Ruminococcus-like microbe was associated with IBS, indicating the potential utility of microbe discovery for gastrointestinal disorders. A greater frequency of pain correlated with an increased abundance of several bacterial taxa from the genus Alistipes. CONCLUSIONS: Using 16S metagenomics by PhyloChip DNA hybridization and deep 454 pyrosequencing, we associated specific microbiome signatures with pediatric IBS. These findings indicate the important association between gastrointestinal microbes and IBS in children; these approaches might be used in diagnosis of functional bowel disorders in pediatric patients.