Uropathogenic Escherichia coli (UPEC) are a major cause of urinary tract and bloodstream infections, and a significant driver of increasing antibiotic resistance. Several globally dominant UPEC lineages have been described, the most common being the multidrug resistant UPEC sequence type 131 (ST131) clone. ST131 was identified in 2008 as a major clone linked to the spread of the CTX-M-15 extended-spectrum β-lactamase (ESBL)-resistance. Since then, ST131 has also been strongly associated with fluoroquinolone resistance. In this presentation, I will discuss our recent work on the molecular characterisation of UPEC ST131, including the use of genome sequencing to demonstrate its rapid and recent global dispersal, and the development of a high-throughput transposon mutagenesis system in combination with next generation sequencing to understand the regulation of key virulence factors and the spread of antibiotic resistance genes.