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The adherent invasive Escherichia coli (AIEC) pathotype is implicated in IBD pathogenesis. AIEC strains are currently defined by phenotypic measurement of their pathogenicity, including invasion of epithelial cells. This broad definition, combined with the genetic diversity of AIEC across IBD patients, has complicated the identification of virulence determinants. Herein, we perform whole genome sequencing of 168 E. coli samples isolated from IBD patients. Using pangenome-wide comparisons of E. coli clinical isolates, we correlate accessory genes that co-segregate with an invasion phenotype. We found the acquisition of antibiotic resistance genes in clinical isolates confounds the traditional gentamicin protection assays used to quantify invasion. We developed an alternative amikacin-based antibiotic protection assay to which none of the clinical isolates were resistant, and used this assay to identify genes that co-segregate with invasion. Our approach using both long read and short read sequencing paired with accurate phenotypic characterization enabled us to identify putative virulence genes in strains of E. coli from IBD patients. Our study helps explain why single genetic markers for AIEC have been challenging to identify and highlights the importance of incorporating antibiotic resistance screening for invasion assays used in AIEC identification.