Anaplasma phagocytophilum, the causative agent of human granulocytotropic anaplasmosis, can infect white-tailed deer (WTD; Odocoileus virginianus), and this species is a crucial host for adult Ixodes scapularis, the primary vector of A. phagocytophilum. The goal of this study was to determine the geographic distribution of A. phagocytophilum among WTD across a 19 state region and to evaluate the utility of WTD as natural sentinels. Serologic testing using the indirect fluorescent antibody (IFA) assay was conducted on WTD serum samples and molecular and xenodiagnostic tests were performed to confirm serologic results. The surveillance system was assessed through examination of vital attributes including WTD age and gender associations with serologic status, sample size adequacy for accurate infection status classification, and presence of the vector, I. scapularis. Six hundred thirty-three of 2,666 (24%) WTD in 17 states tested positive for antibodies (>or=128) when tested by IFA assay. Testing for p44 and/or 16S rRNA gene targets identified 73 (16%) PCR positive WTD among 458 animals tested, all of which originated from seropositive populations. Attempts to culture A. phagocytophilum from WTD were unsuccessful; however, xenodiagnostic mice inoculated with blood from 3 WTD became infected. Seroprevalence did not differ by deer age or gender; however, WTD<or=0.75 years old had a higher prevalence of PCR positivity. Using seroprevalence data, a sample size of 6-9 animals per population was projected to be adequate for identifying seropositive populations. The presence of I. scapularis was significantly associated with A. phagocytophilum antibodies in WTD. Collectively, the results of this study demonstrate that WTD would be suitable natural sentinels for this emerging zoonotic pathogen.