Background: The rising prevalence of Extended-spectrum β-lactamase-producing (ESBL) Escherichia coli poses a significant threat to human and animal health. Methods: To address this, we conducted a longitudinal two-year One Health study to assess ESBL E. coli occurrence and distribution across dairy farms, surrounding environments, and urban wastewater in a peri-urban region of Western Canada. Results: A total of 546 presumptive ESBL E. coli were recovered, with the highest occurrence in wastewater influent (75.9%) and calf feces (73.6%), and lowest in soil (6.3%) and surface water (18.8%). Seasonal analysis showed a significantly higher occurrence in summer compared to spring. The bla_CTX-M-15 gene predominated (79%), followed by bla_TEM (28%) and bla_SHV (9%), with most isolates harboring multiple ESBL genes. Whole-genome sequencing of 387 isolates identified 75 resistance determinants spanning nine antimicrobial classes, including 24 β-lactamase genes and 10 CTX-M variants. Ninety-four sequence types (STs), including nine novel STs, were detected. The most common STs were ST648, ST69, and ST10, with distinct distributions across sources. Plasmid analysis revealed extensive diversity, with approximately half of the plasmid types shared across multiple sample types, indicating potential horizontal gene transfer. Over 200 virulence factors were identified, including toxin genes and Shiga toxin-associated genes, primarily in calf and surface water isolates. Phylogroups A and B1 dominated samples from dairy farms, and phylogroup B2 was restricted to wastewater and surface water. Conclusions: These findings identify the environment as a reservoir for ESBL E. coli and reveal the unexpected predominance of the emerging MDR ST648 lineage, rather than ST131, and reinforce the need for comprehensive integrated One Health surveillance.