Escherichia coli sequence types (ST) 69, 73, 95 and 131 are collectively responsible for a large proportion of E. coli urinary tract and bloodstream infections, and differ markedly in their antibiotic susceptibility. We describe a novel PCR method to detect and distinguish these lineages rapidly. Three hundred and eighteen published E. coli genomes were compared, to identify signature sequences unique to each of the four major STs. The specificity of these sequences was assessed in silico by seeking them in an additional 98 genomes. A PCR assay was designed to amplify size-distinguishable fragments unique to the four lineages, and was validated using 515 E. coli isolates of known ST. Genome comparisons identified 22 regions, ranging in size from 335 bp to 26.5 kb, unique to one or other of the four predominant E. coli STs, with two to ten specific regions per ST. These regions predominantly harbored genes encoding hypothetical proteins and were within or adjacent to prophage sequences. Most (13/22) were highly conserved (>96.5% identity) in genomes of the respective ST. The new assay identified correctly all of 142 representatives of the four major STs in the validation set (n=515), with only two ST12 isolates misidentified as ST95. Compared with MLST, the assay thus had 100% sensitivity and 99.5% specificity. Rapid identification of major extra-intestinal E. coli STs will benefit future epidemiological studies and could be developed to tailor antibiotic therapy to the different susceptibilities of these dominant lineages.