Analysis of Escherichia coli taxonomy has expanded into a species-complex with the identification of divergent cryptic clades. A key question is the evolutionary trajectory of these clades and their relationship to isolates of clinical or veterinary importance. Since they have some environmental association, we screened a collection of E. coli isolated from a long-term spring barley field trial for their presence. While most isolates clustered into the enteric-clade, four of them clustered into Clade-V, and one in Clade-IV. The Clade -V isolates shared >96% intra-clade average nucleotide sequence identity but <91% with other clades. Although pan-genomics analysis confirmed their taxonomy as Clade -V (E. marmotae), retrospective phylogroup PCR did not discriminate them correctly. Differences in metabolic and adherence gene alleles occurred in the Clade -V isolates compared to E. coli sensu scricto. They also encoded the bacteriophage phage-associated cyto-lethal distending toxin (CDT) and antimicrobial resistance (AMR) genes, including an ESBL, blaOXA-453. Thus, the isolate collection encompassed a genetic diversity, and included cryptic clade isolates that encode potential virulence factors. The analysis has determined the phylogenetic relationship of cryptic clade isolates with E. coli sensu scricto and indicates a potential for horizontal transfer of virulence factors.