Although nine nominal species of Eimeria Schneider, 1875 have been reported from owls (Strigiformes Wagler), molecular data are available for only one, Eimeria megabubonis Upton, Campbell, Weigel & McKown, 1990. This species has been reported from the great horned owl, Bubo virginianus (Gmelin) at the type-locality of Kansas, USA, and the additional localities of Oklahoma and Mississippi, USA. Eimeria bubonis Cawthorn & Stockdale, 1981, also from B. virginianus, is represented in the literature only by its original description from Saskatchewan, Canada. The present work reports a novel host locality of E. bubonis from B. virginianus in Arkansas, USA. Additionally, novel molecular data from the 18S ribosomal RNA gene and the cytochrome c oxidase subunit 1 mitochondrial gene is provided for E. bubonis. Phylogenetic analysis using the newly generated and previously published sequence data for Eimeria spp. is also presented.