BackgroundLyme disease can affect people, dogs, and horses, but it remains poorly understood, especially in the horse. Determining the seroprevalence of Borrelia burgdorferi in horses in different geographic areas will enable better understanding of the epidemiology of the disease, thus improving diagnosis and treatment of affected animals.HypothesisTo determine the seroprevalence of B. burgdorferi in horses in southwest Virginia.AnimalsHorses presented for routine Coggins testing from January 2013 to January 2014 had additional blood drawn for Lyme Multiplex Assay testing.MethodsOf 492 samples collected, 250 samples were analyzed using the Lyme Multiplex Assay. Of the 83 horses that had positive test results to at least 1 outer surface protein (Osp), 63 were available for follow‐up testing 5–17 months later (June 2014).ResultsThirty‐three percent of horses had positive results for antibodies to at least 1 Osp. Horses with a positive outer surface protein F (OspF) result were older (14.5 ± 0.79) than horses with a negative OspF result (11.6 ± 0.53). Of the horses available for follow‐up testing, 63% had the same result as that of the initial test. There was no difference in test result between initial and follow‐up testing.ConclusionsHorses seropositive to B. burgdorferi are common in Virginia, and older horses are more likely to have a positive test result for OspF than younger horses. Follow‐up testing indicated that the majority of horses that were positive on initial testing did not have a different test result 5–17 months later.