During the fall of 2010, 332 deer serum samples were collected from 15 of the 16 (93.8%) Maine counties and screened for eastern equine encephalitis virus (EEEV) antibodies using plaque reduction neutralizing tests (PRNTs). The aim was to detect and map EEEV activity in the state of Maine. Forty-seven of the 332 (14.2%) sera were positive for EEEV antibodies, showing a much wider distribution of EEEV activity in Maine than previously known. The percentage of EEEV antibody-positive deer sera was ≥10% in six counties-Piscataquis (100%), Somerset (28.6%), Waldo (22.2%), Penobscot (21.7%), Kennebec (13.7%), and Sagadahoc (10%). Positive sera were detected in all the six counties (Somerset, Waldo, Penobscot, Kennebec, Cumberland, and York) that were positive in 2009, suggesting endemic EEEV activity in these counties. EEEV antibodies were not detected in sera collected in five counties-Franklin, Knox, Lincoln, Oxford, and Washington-which was either due to low sample size or lack of EEEV activity in these counties. Our data suggest higher EEEV activity in central Maine compared to southern Maine, whereas EEEV activity in Maine has historically been associated with the southern counties of York and Cumberland.