Extra-intestinal pathogenic Escherichia coli (ExPEC) are a significant cause of urinary tract infections and bacteraemia in the UK and around the world. These E. coli primarily belong to phylogenetic groups B2 and D, with the clones ST131, ST127, ST95, ST73 and ST69 responsible for the majority of these infections. In the UK, studies of ExPEC have focused on isolates from the North of England, ST131 strains and ExPEC that possess extended-spectrum beta-lactamase (ESBL) enzymes. Therefore, very little is understood about the UK ExPEC population as a whole, the breadth of virulence factors contributing to these infections and the differences between urinary and bloodstream-derived ExPEC. In this study ST131 was more frequently detected in bloodstream isolates and ST95 was most prevalent in urinary isolates. Comparative virulence of the major clones in the Galleria mellonella infection model revealed ST131 isolates to effect the highest mortality, although serogroup O6, which is linked with ST73, was also associated with high mortality, potentially explaining the success of ST73-O6 in bacteraemia. Analysis of virulence factors identified pap, afa/dra and kpsMTII as important determinants in isolates causing urosepsis and those of ST131, while fyuA and fimH were distinctly lacking, demonstrating their role as colonisation factors rather than virulence factors. Although these findings are important, with appropriate treatment of urinary tract infections they can become redundant, as ExPEC would be eradicated before causing a severe infection such as bacteraemia or urosepsis. In urinary isolates, resistance to trimethoprim approached 50% and ampicillin resistance was >70%, while ST131 isolates as a whole demonstrated ciprofloxacin and trimethoprim resistance >50%. Together these indicate that empirical UTI guidelines need to be revisited, to prevent recurrence of infection and ascension to the kidneys and bloodstream. In addition, data from this study can be used to develop a point-of-care test to detect ST131, to guide appropriate treatment, without the delay associated with referring a urine specimen for microbiological investigation.