In this study, we investigated the population structure of extended-spectrum beta-lactamase (ESBL)-producing Escherichia coli in Spain and determined possible associations between specific multilocus sequence typing (MLST) types and ESBL types. Ninety-two ESBL-producing E. coli isolates from 11 Spanish hospitals were studied. The predominant ESBLs in this collection were CTX-M-14 (45.7%), SHV-12 (21.7%) and CTX-M-9 (20.6%). Phylogenetic groups and MLST types were studied. Thirty-seven isolates (40.2%) belonged to phylogroup A, 26 (28.3%) to group B1, 13 (14.1%) to group B2 and 16 (17.4%) to group D. Fifty-six sequence types (STs) were identified, but ST131 (eight isolates) and ST167 (five isolates) were the most prevalent. The most common ST complexes were ST10 (13 isolates; 14.3%) and ST23 (10 isolates; 11%). Escherichia coli ST131 carried six different ESBLs (CTX-M-1, CTX-M-9, CTX-M-10, CTX-M-14, CTX-M-15 and SHV-12), E. coli ST10 complex carried five ESBLs and E. coli ST23 complex carried four ESBLs. A great diversity of MLST types was observed among Spanish ESBL-producing E. coli isolates.