In order to better understand the ecological and virulence characteristics of the various clades of Escherichia, in vitro and in vivo experiments were undertaken. Members of the recently described cryptic clades of Escherichia (clades III, IV, and V) were found to have an enhanced ability to form biofilms compared to strains of Escherichia coli, E. fergusonii, or E. albertii. Members of the cryptic clades were also able to replicate at a lower temperature (5°C versus 11°C) than strains of the named species of Escherichia. Neither a strain's maximal growth rate nor its optimal temperature for growth varied with respect to the strain's phylogenetic affiliation. Escherichia strains not belonging to the species E. coli were positive for a mix of traits thought to enhance a strain's ability to cause either intestinal or extraintestinal disease. However, no non-E. coli Escherichia strain was virulent in a mouse model of extraintestinal infection. The frequency of resistance to antibiotics was low, and none of the strains tested harbored class 1, 2, or 3 integrons. The results of these experiments support the hypothesis that members of the cryptic Escherichia clades may be better able to persist in the external environment compared to E. coli, E. fergusonii, or E. albertii, isolates.