Invasive Escherichia coli disease (IED) is associated with high hospitalization and mortality rates, particularly among adults aged ≥60 years. O-antigens are virulence factors required for E. coli survival. To inform EXPEC9V development, a novel glycoconjugate vaccine targeting E. coli O-antigens that is no longer in active clinical development, this retrospective observational study describes O-serotype prevalence among E. coli isolates from IED patients. Eligible patients were identified from medical record databases (9 January 2018–8 November 2019) across 17 tertiary care hospitals in Europe, North America, and Asia. To estimate vaccine serotype coverage of EXPEC9V, E. coli isolates were O-serotyped using whole-genome sequencing and agglutination. Antimicrobial susceptibility testing was also performed. Nine hundred and two patients were enrolled, of whom 690 (76.5%) were aged ≥60 years. Common serotypes were O25, O2, O6, O1, O15, O75, O16, O4, and O18, with O25 being the most reported (17.3%). In patients aged ≥60 years, 422/637 E. coli isolates were EXPEC9V O-serotypes. EXPEC9V O-serotype prevalence did not substantially differ when stratified according to sex, presence of a positive blood culture, sepsis, fatality, or multidrug resistance. Consistent with previous studies, serotype O25 was most prevalent and associated with ~20% of cases. An EXPEC9V vaccine serotype coverage of 66.2% was observed for IED patients aged ≥60 years.