The objective of this work was to search for potential wildlife reservoir hosts of Encephalomyocarditis virus (EMCV) in wildlife species. From 1994 to 2006, 317 blood and tissue samples were collected from 17 wildlife species in Greece. Encephalomyocarditis virus was isolated only from two Rattus rattus. In addition, antibody to EMCV were detected in sera from 39 Rattus rattus, one Apodemus sylvaticus, one Microtus arvalis, and 13 Sus scrofa.