Enterohaemolysin (Ehx) and alpha-haemolysin are virulence-associated factors (VAFs) causing the haemolytic phenotype inEscherichia coli. It has been shown that chromosomally and plasmid-encoded alpha-haemolysin are characteristic of specific pathotypes, virulence-associated factors and hosts. However, the prevalence of alpha- and enterohaemolysin does not overlap in the majority of pathotypes. Therefore, this study focuses on the characterization of the haemolyticE. colipopulation associated with multiple pathotypes in human and animal infectious diseases. Using a genomics approach, we investigated characteristic features of the enterohaemolysin-encoding strains to identify factors differentiating enterohaemolysin-positive from alpha-haemolysin-positiveE. colipopulations. To shed light on the functionality of Ehx subtypes, we analysed Ehx-coding genes and inferred EhxA phylogeny. The two haemolysins are associated with a different repertoire of adhesins, iron acquisition or toxin systems. Alpha-haemolysin is predominantly found in uropathogenicE. coli(UPEC) and predicted to be chromosomally encoded, or nonpathogenic and undeterminedE. colipathotypes and typically predicted to be plasmid-encoded. Enterohaemolysin is mainly associated with Shiga toxin-producingE. coli(STEC) and enterohaemorrhagicE. coli(EHEC) and predicted to be plasmid-encoded. Both types of haemolysin are found in atypical enteropathogenicE. coli(aEPEC). Moreover, we identified a new EhxA subtype present exclusively in genomes with VAFs characteristic of nonpathogenicE. coli. This study reveals a complex relationship between haemolyticE. coliof diverse pathotypes, providing a framework for understanding the potential role of haemolysin in pathogenesis.