: Escherichia coli classification into phylogroups reflects the diversity of their pathogenicity and their ecological niche, B2 isolates being the most virulent among extra-intestinal strains. MALDI-TOF MS allows a quick, automated, simple and inexpensive bacterial identification. We evaluated the MALDI-TOF MS as a tool for E. coli phylogroup differentiation. We used 656 E. coli isolates, previously assigned to phylogroup A, B1, B2, and D by multiplex PCR, to constitute independent training and validation sets. We then defined two phylogrouping strategies, both validated on spectra obtained by the 'direct transfer method'. The first strategy used the MALDI Biotyper software (Bruker Daltonik) that identified a single peak shift between isolates of phylogroup B2 and those of groups A, B1 and D. It accurately classified 89% of the isolates. The second strategy used the ClinProTools software (Bruker Daltonik) and was based on three successive models. The model 1 adequately differentiated 92% of phylogroup B2-isolates from those belonging to phylogroups A, B1, D. The model 2 adequately discriminated 87% of phylogroup D-isolates from those of phylogroups A and B1. The model 3 correctly sorted 69% of A and B1-isolates. We concluded that clinical laboratories could routinely and very quickly assign E. coli isolates to phylogroups with MALDI-TOF MS. These methods could (i) expedite the detection of the most virulent strains belonging to phylogroup B2 and (ii) be a first-line tool to monitor the epidemiology of extra-intestinal pathogenic E. coli.