In this paper, we demonstrate that Fourier transform infrared (FT-IR) spectroscopy is able to discriminate rapidly between uropathogenic Escherichia coli (UPEC) of key lineages with only relatively simple sample preparation. A total of 95 bacteria from six different epidemiologically important multilocus sequence types (ST10, ST69, ST95, ST73, ST127 and ST131) were used in this project and principal component-discriminant function analysis (PC-DFA) of these samples produced clear separate clustering of isolates, based on the ST.