Extraintestinal pathogenic E. coli (ExPEC) sequence type 95 (ST95) is one of the top five pandemic ExPEC lineages. It causes severe disease in humans and poultry but the understanding of its phylogenetic structure and the unique features of its sublineages have remained limited. Here a core genome analysis of 668 ST95 isolates identified 10 clades (A to J) of which five are reported here for the first time. Analysis of F plasmid carriage showed that almost a third (178/668 [27%]) of the collection carry pUTI89 (F29:B10), an ExPEC virulence plasmid phylogenetically restricted to human sourced E. coli from clade A and one sublineage of clade B. In contrast, multiple ST95 clades comprising almost half (328/668 [49%]) of the collection harbor ColV plasmids with multiple F types. Strikingly, unlike isolates carrying pUTI89, ST95 lineages with ColV were sourced from poultry and humans. An analysis of a cohort of 34,176 E. coli isolates comprising 2,570 sequence types mirrored what we observed in ST95: (i) pUTI89 was overwhelmingly linked to E. coli sourced from humans but almost entirely absent from 13,027 E. coli isolates recovered from poultry, pigs, and cattle, and (ii) E. coli isolates harboring ColV plasmids were from multiple sources, including humans, poultry, and swine. Overall, our data suggest that F plasmids influence E. coli host range, clade structure, and zoonotic potential in ST95 and ExPEC more broadly. Our study also indicates that the role of food animals as a source of human ExPEC disease is complex and warrants further investigation.