Extended-spectrum beta-lactamase (ESBL)-producing extraintestinal pathogenic Escherichia coli (ExPEC), particularly high-risk lineages, are responsible for severe infections and increased mortality and hospital costs worldwide, with a major burden in low-income countries. Here we determined the antimicrobial susceptibility and performed whole-genome sequencing of E. coli isolates from extraintestinal infections of patients during 2017-2018 at Maputo Central Hospital (Mozambique). Multidrug resistance was displayed by 71% of isolates (17/24). All isolates resistant to cefotaxime and ceftazidime were positive for ESBL genes (16/24; 67%) and were co-resistant to amoxicillin/clavulanate (14/16; 88%), piperacillin/tazobactam (8/16; 50%), gentamicin (12/16; 75%), trimethoprim/sulfamethoxazole (15/16; 94%) and ciprofloxacin (11/16; 69%). Several major high-risk ExPEC lineages were identified, such as H 30RxST131, fim H41-ST131, H 24Rx-ST410, ST617, ST361 and ST69 harbouring bla CTX-M-15 , and H 30R-ST131, ST38 and ST457 carrying bla CTX-M-27 . Dissemination of CTX-M transposition units (IS Ecp1 -bla CTX-M-15 -orf477 and IS Ecp1 -bla CTX-M-27 -IS 903B ) among different sequence types could be occurring through the mobility of IncF plasmids. Additionally, all H 24Rx-ST410 isolates carried IS Ecp1 -mediated bla CMY-2 AmpC and specific mutations in PBP3/OmpC proteins, potentially contributing to carbapenem resistance even in the absence of carbapenemase genes. Genome analysis highlighted a high assortment of ExPEC/UPEC virulenceassociated genes mainly involved in adhesion, invasion, iron uptake and secretory systems among isolates, and an ExPEC/EAEC hybrid pathotype ( fimH 27-ST131_O18-ac:H4) showing the highest virulence gene content. cgMLST showed clonality and closely related isolates, particularly among ST131 and ST410, suggesting hospital-acquired infections and long-term ward persistence. Our study provides new insights into ExPEC clones, urging measures to prevent and contain their diffusion in this hospital and Mozambique. (c) 2022 Elsevier Ltd and International Society of Antimicrobial Chemotherapy. All rights reserved.