We aimed to develop a reliable method based on Fourier transform infrared spectroscopy with attenuated total reflectance (FTIR-ATR) to discriminate Escherichia coli clones from B2(n = 9) and D(n = 13) phylogenetic groups. Eighty-eight E. coli isolates belonging to phylogenetic groups B2(n = 39) and D(n = 49), including particularly widespread high risk clones or clonal complexes (HiRCC) ST131, ST69, ST393 and ST405 were studied. Spectra were analysed by unsupervised (hierarchical cluster analysis-HCA) and supervised methods (soft independent modelling of class analogy-SIMCA and partial least square discriminant analysis-PLSDA). B2-ST131 isolates were discriminated from B2 non-ST131 and D phylogroup isolates (ST69, ST393, ST405) by HCA, SIMCA and PLSDA. D-ST69, D-ST393 and D-ST405 isolates were also distinguished from each other and from other STs from phylogroup D by the three methods. We demonstrate that FTIR-ATR coupled with chemometrics is a reliable and alternative method to accurately discriminate particular E. coli clones. Its validation towards an application at a routine basis could revolutionize high-throughput bacterial typing.