Extra-intestinal pathogenic Escherichia coli (ExPEC) are a significant cause of urinary tract infections and bacteraemia worldwide. Currently no single virulence factor or ExPEC lineage has been identified as the sole contributor to severe extra-intestinal infection and/or urosepsis. Galleria mellonella has recently been established as a simple model for studying the comparative virulence of ExPEC. In this study we investigated the virulence of 40 well-characterized ExPEC strains, in G. mellonella, by measuring mortality (larvae survival), immune recognition/response (melanin production) and cell damage (lactate dehydrogenase production). Although mortality was similar between urinary and bloodstream isolates, it was heightened for community-associated infections, complicated UTIs and urinary-source bacteraemia. Isolates of ST131 and those possessing afa/dra, ompT and serogroup O6 were also associated with heightened virulence.