Escherichia coli is one of the major pathogens responsible for bactaeremia. Empirical antibiotherapy of these infections usually relies on third-generation cephalosporins (3GCs). Thus, the occurrence and epidemiology of 3GC-resistant strains have to be monitored. The French prospective multicentre study COLIBAFI collected 1081 strains of E. coli responsible for bacteraemia in 2005. In the present work, the prevalence of resistance to 3GCs was evaluated, and the implicated molecular mechanisms were characterized by specific PCR and sequencing. Phylogenetic grouping, O-typing, pulsed-field gel electrophoresis and virulence factor analysis were used to investigate the genetic background of the 3GC-resistant (3GC-R) strains. Clinical features of the patients with documented data (n = 1051) were analysed. Decreased susceptibility to 3GCs was observed in 41 strains (3.8%): 19, 18 and four had extended-spectrum β-lactamase (ESBL), AmpC cephalosporinase and OXA-type penicillinase phenotypes, respectively. Pulsed-field gel electrophoresis revealed that the 3GC-R strains constitute a diverse population. All but one of the strains with an ESBL phenotype produced a CTX-M-type enzyme, and six of them belonged to the widespread intercontinental clone O25b:H4-ST131. AmpC phenotype strains harboured various chromosomal ampC promoter and coding region mutations and/or the bla(CMY-2) plasmidic gene. 3GC-R strains carried fewer virulence factors and were more co-resistant to other antibiotics than 3GC-susceptible (3GC-S) strains. Infections with 3GC-R strains were mostly community-acquired and, as compared with those caused by their 3GC-S counterparts, were more severe. Underlying chronic disease and prior use of antibiotics were independent risk factors for development of a 3GC-R strain bacteraemia. The fact that the molecular support of 3GC resistance is mainly plasmid-mediated represents a potentially epidemic threat.