This study presents a comprehensive approach of a clonal diversity analysis of 448 Extended-spectrum beta-lactamase (ESBL)-producing E. coli isolated from environmental, human and food samples in Spain. The phenotypic confirmation of ESBL production was performed by disc diffusion and microdilution methods, while Polymerase Chain Reaction (PCR) and sequencing were used for the molecular characterization of beta-lactamase genes (bla(CTX-M), bla(SHV), bla(TEM), bla(OXA)). Clonal relationship of isolates was determined by multi-locus sequence typing (MLST). Multidrug resistant strains were present in all the studied niches, with percentages above 50.0%. The most prevalent beta-lactamase genes were bla(CTXM-14) (26%) and bla(CTXM-1) (21.4%), followed by bla(SHV-12), bla(CTX-M-15) and bla(TEM-42). MLST isolates were grouped into 26 clonal complexes (CC) and 177 different sequence types (ST) were detected. Despite the high clonal diversity observed, CC10 was the prevalent and the only CC detected in all niches, while other complexes as CC131 were mainly associated to human isolates. The observed prevalence and diversity of these resistant bacteria across the different environments encourages a One Health approach to prevent and control ESBL dissemination between environment and consumers.