We used PCR and a novel serologic assay to determine infection and exposure rates to Ehrlichia ewingii in dogs from an area of northeast Oklahoma and northwest Arkansas where Amblyomma americanum ticks are abundant. Of 143 dogs assayed, 13 (9.1%) harbored E. ewingii by PCR and 64 (44.8%) had antibodies to E. ewingii detected using a peptide-based microtiter plate ELISA. Dogs were more likely (P=0.001) to be positive by PCR if sampled in August (30.8%) but no association was found between seropositive status and month of collection of sample (P>0.05). Additional testing revealed PCR evidence of Ehrlichia chaffeensis (4/143; 2.8%) and Anaplasma platys (5/143; 3.5%) as well as antibodies reactive to E. chaffeensis (25/143; 17.5%), Ehrlichia canis (2/143; 1.4%), and Anaplasma spp. (8/143; 5.6%). Testing of another 200 dogs from the area revealed additional PCR and/or serologic evidence of E. ewingii, E. canis, E. chaffeensis, and A. platys. None of the 343 dogs evaluated had evidence of Borrelia burgdorferi exposure. These data support the interpretation that E. ewingii may be the primary agent of canine ehrlichiosis in this region, and suggest that diagnostic evaluation of dogs suspected to have a tick-borne disease should include assays targeting this organism.