In recent years, several species of ehrlichiae have been recognized as tick-borne disease agents of veterinary and medical importance. Clinically normal free-ranging or previously free-ranging lemurs, including 46 ring-tailed lemurs (Lemur catta), six blue-eyed black lemurs (Eulemur macaco flavifrons), and four black and white ruffed lemurs (Varecia variegata variegata) from St. Catherines Island, Georgia, were tested for evidence of exposure to tick-borne ehrlichiae. All 52 adult lemurs were serologically tested for exposure to Ehrlichia chaffeensis and Anaplasma phagocytophilum. Polymerase chain reaction (PCR) assays for E. chaffeensis, A. phagocytophilum, Ehrlichia ewingii, and Ehrlichia canis were conducted on blood samples from all 56 lemurs. Blood from all lemurs was inoculated into DH82 cell cultures for E. chaffeensis isolation. Of the adult lemurs, 20 (38.5%) and 16 (30.8%) had antibodies reactive (≥1:128) for E. chaffeensis and A. phagocytophilum, respectively. Two ring-tailed lemurs were PCR and culture positive for E. chaffeensis. Molecular characterization of the two E. chaffeensis isolates showed that both contained 5-repeat variants of the variable-length PCR target (VLPT) antigen gene and 3-repeat variants of the 120-kDa antigen gene. Sequencing of the VLPT genes revealed a novel amino acid repeat unit (type-9). One lemur infected with E. chaffeensis was slightly hypoproteinemic and had moderately elevated serum alanine aminotransferase (ALT) levels. These lemurs from St. Catherines Island have been exposed to or infected with tick-borne ehrlichiae, or both, but showed no clinical disease.