Avian pathogenic Escherichia coli (APEC) cause respiratory and systemic disease in poultry. Sequencing of a multi-locus sequence type (ST)-95 serogroup O1 strain previously indicated that APEC resemble E. coli causing human extra-intestinal diseases. We sequenced the genomes of two strains of another dominant APEC lineage (ST23 serogroup O78 strains χ7122 and IMT2125) and compared them to each other and the reannotated APEC O1 sequence. For comparison, we also sequenced a human enterotoxigenic E. coli (ETEC) strain of the same ST23 serogroup O78 lineage. Phylogenetic analysis indicated that the APEC O78 strains were more closely related to a human ST23 ETEC than APEC O1, indicating that separation of pathotypes on the basis of being extra-intestinal or diarrheagenic is not supported by their phylogeny. The accessory genome of APEC ST23 strains exhibited limited conservation of APEC O1 genomic islands and a distinct repertoire of virulence-associated loci. In light of this diversity, we surveyed the phenotype of 2185 χ7122 signature-tagged transposon mutants following intra-air sac inoculation of turkeys. This identified novel APEC ST23 genes that play strain- and tissue-specific roles during infection. For example, genes mediating group 4 capsule synthesis were required for virulence of χ7122 and conserved in IMT2125 but absent from APEC O1. Our data reveal the genetic diversity of E. coli adapted to cause the same avian disease and indicate that the core genome of the ST23 lineage serves as a chassis for evolution of E. coli adapted to cause avian or human disease via acquisition of distinct virulence genes.