Several recent studies have highlighted the emergence of a globally disseminated clone of uropathogenic and invasive Escherichia coli isolates of serotype O25:H4 and sequence type 131. The ability to characterize rapidly E. coli isolates of this lineage would facilitate enhanced surveillance for this pathogen. We have used the semi-automated DiversiLab repetitive PCR-based system to analyse a collection of 35 clinical isolates of uropathogenic E. coli from across the UK, with particular focus on the O25:H4-ST131 lineage. All isolates had been characterized using multilocus sequence typing (MLST), and 14 had previously been typed using pulsed-field gel electrophoresis (PFGE). The DiversiLab system allowed discrimination of O25:H4-ST131 isolates from those of other E. coli lineages. It was slightly more discriminatory than MLST, but was less discriminatory than PFGE. With an analysis time of <4 h between receipt of a cultured organism and provision of a typing result, the system offers information on a real-time basis, a major advantage over current practice. We suggest that introduction of the DiversiLab system would be useful for rapid exclusion of E. coli isolates during outbreak investigations, and that the approach could be employed for surveillance for pathogenic or antibiotic-resistant clones of this organism.