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Third-generation cephalosporin resistance (3GC-R) in Escherichia coli is a rising problem in human and farmed-animal populations. We conducted whole-genome sequencing analysis of representative 3GC-R isolates previously collected from dairy farms in southwest England, and human urinary isolates collected from the same geographical area and time-period, and confirmed by PCR to carry acquired 3GC-R genes. This analysis identified blaCTX-M (131 isolates encoding CTX-M-1, -14, -15, -and 32 and the novel variant CTX-M-214), blaCMY-2 (6 isolates), and blaDHA-1 (1 isolate). A highly conserved plasmid was identified in 73 isolates, representing 27 E. coli sequence types. This novel ∼220-kb IncHI2 plasmid carrying blaCTX-M-32 was sequenced to closure and designated pMOO-32. 'This data is NERC-funded but not held by the EIDC. This data is archived in the European Nucleotide Archive'.