Aims: The aim of this study was to characterize Escherichia fergusonii and Escherichia albertii isolated from water. Methods and Results: The characterization of E. fergusonii and E. albertii isolated from water was determined using an Escherichia coli-specific uidA PCR, a tuf PCR, and with phylogenetic analysis using three housekeeping genes (adk, gyrB, and recA) from the E. coli MLST scheme, selected for their ability to discriminate among all Escherichia species. Among the 527 isolates tested, 25 (4.7%) were uidA PCR negative and tuf PCR positive. Phylogenetic analysis using adk, gyrB and recA genes showed that 6, 18 and 1 of these 25 non-E. coli Escherichia spp. isolates grouped with reference strains of E. fergusonii, E. albertii, and E. coli, respectively. Finally, the 25 non-E. coli Escherichia spp. strains isolated were investigated for the presence of pathogenic factors, comprising intimin (eae gene), cytolethal distending toxin (cdtB gene) and shiga toxin (stx gene). With the PCR primers used, the presence of eae and stx genes was not detected. However, cdtB genes types I/IV were detected for 3 (16.7%) E. albertii strains, whereas 15 of 18 (83.3%) possessed the cdtB gene types II/III/V. Conclusions: These results showed that MLST scheme allows a more accurate identification of non-E. coli species than phenotypic tests. We also showed that E. fergusonii and E. albertii represent, respectively, 0.8 and 2.5% of all Escherichia species isolated and the pathogenic cdtB genes were present in 83.3% of these strains. Significance and impact of the study: The data presented in this study provided an efficient way to correctly identify non-E. coli species contributing to our understanding of the risks associated with Escherichia species in water consumed by humans and animals. Furthermore, the results give an insight about the natural habitats of these species.