During the winter of 2004, 48 fecal samples were collected from live-trapped fox squirrels (Sciurus niger) from central Wyoming (Natrona County) and examined for species of Eimeria. Two species, Eimeria lancasterensis (prevalence, 65%) and Eimeria ontarioensis (prevalence, 27%), were identified. Genomic DNA sequences ITS1 and ITS2 were amplified, cloned, and sequenced. Additional sequences from E. lancasterensis isolated from a Delmarva fox squirrel (Sciurus niger cinereus) collected on Chincoteague Island, Virginia, were also identified. Comparison of pairwise distances suggests that E. lancasterensis from Wyoming and Virginia are conspecific. Maximum Parsimony tree construction identified 2 lineages, one E. ontarioensis and one E. lancasterensis; and both lineages had a strong bootstrap support (100%). The Maximum Parsimony analysis was unable to resolve the Wyoming and Virginia strains.