Eastern equine encephalitis virus (EEEV) is highly pathogenic, mosquito-borne, and capable of causing disease and mortality in birds, humans, and other mammals. This study described 3 cases of EEEV in puppies in Michigan and New York in 2011. The puppies all had a history of acute fever and diarrhea. One died in its kennel overnight, and the other 2 developed seizures and were euthanized. Two puppies tested negative for West Nile virus and canine distemper virus; one of these puppies also tested negative for toxoplasmosis. All 3 study puppies tested negative for rabies. Histologic examination revealed necrotizing meningoencephalitis with positive immunohistochemical labeling for EEEV antigen. Diagnosis was confirmed through PCR or plaque reduction neutralization testing. These cases occurred in late summer in areas with high mosquito activity. EEEV should be considered as a differential diagnosis in puppies with neurologic signs, particularly if more common differentials (eg, rabies, canine distemper, toxoplasmosis) have been ruled out.