Serum from 226 free-ranging white-tailed deer (Odocoileus virginianus) was screened for Eastern Equine Encephalitis Virus (EEEV) antibodies using plaque reduction neutralization tests. EEEV antibodies were detected in 7.1% of samples. This is the first time EEEV antibodies have been detected in O. virginianus populations in the state of Maine (ME). The highest percentage of EEEV positive sera was in Somerset County (19%) in central ME, and this is the first time that EEEV activity has been detected in that County. EEEV RNA was not detected in any of the 150 harvested deer brain samples submitted to the ME Department of Inland Fisheries and Wildlife as a part of screening for Chronic Wasting Disease. This suggests that screening deer brains is not an efficient method to detect EEEV activity. For each serum sample tested, the geographic location in which the deer was harvested was recorded. Significant spatial clustering of antibody-positive sera samples was not detected. Relative to seronegative deer, seropositive deer were slightly more likely to be harvested in nonforested areas compared with forested areas. Results indicate that screening of free-ranging deer sera can be a useful tool for detecting EEEV activity in ME and other parts of North America.