ColicinV (ColV)-like plasmids (ColVLPs) are incompatibility (Inc) F plasmids frequently isolated from human and avian extra-intestinal pathogenic E. coli (ExPEC). ColVLPs encode virulence determinants involved in immune evasion/metal uptake and are strongly linked to increased zoonotic potential. Most ColVLP virulence genes have homologs within chromosomal genomic islands, which we show to be phylogenetically distinct, suggesting divergent function(s) between the ColVLP- and chromosomal-associated copies. To highlight the impacts of such divergence on pathogenesis, we characterised the outer membrane protease OmpT, which is encoded on both ColVLPs (OmpTp) and the chromosome (OmpT) and is associated with increased antimicrobial peptide (AMP) resistance. Our results reveal that the distinct evolutionary pathways of these homologs (~73% AA identity) correspond to differences in enzyme activity. Using an AMP survival assay, we further demonstrated that OmpTp contributes to AMP resistance, where maximal resistance occurs with both OmpTp and OmpT present. Our work also identified several mcr-1+ (enabling resistance to last-line colistin) IncHI2/ColVLP hybrid plasmids. To understand the evolution of these hybrid plasmids, we mapped replicon co-carriage in a draft genome database of 10,000 strains, revealing strong associations between ColVLPs and IncI1, IncX, IncB/O/K/Z, and IncHreplicons. We further identified a series of ColVLP hybrids containing additional IncH, IncN, IncX, IncR, or IncI1 replicons from ExPEC isolates that carried significantly more resistance genes than non-hybrids. Overall, our work: (i) revealed functional differences between ColVLP- and chromosomal-OmpT variants, suggesting that pathogenicity adapted changes may also alter other ColVLP virulence genes to increase ExPEC virulence, and (ii) identified a group of high-risk ColVLP hybrids in ExPEC with the potential to confer both significantly increased antibiotic resistance and enhanced virulence.