The purposes of this study were to estimate the seroprevalence and distribution of horse piroplasmosis, to evaluate risk factors associated with the occurrence of the disease and to compare the different diagnostic methods used for this disease. A total of 253 clinically normal horses were sampled, and a collection form was completed for each horse from five of six different climatic zones of Jordan. The sixth zone was not sampled because it did not include horse population. Competitive enzyme-linked immunosorbent assay (cELISA) revealed 37 horses (14.6%) positive for Theileria equi, and none of the horses was positive for Babesia caballi. Microscopic examination of thin blood smears and PCR test revealed no positive results for either parasite. Grazing was the only risk factor that was associated with being seropositive to the disease; horses that graze are 11.5 more likely to be seropositive (P < 0.05, OR = 11.5, CI: 3.292, 39.962). This is the first study to estimate the prevalence of horse babesiosis using serological test and to identify risk factors associated with the disease in Jordan. Competitive enzyme-linked immunosorbent assay (cELISA) test appears to be more reliable than microscopic examination and PCR in estimating the seroprevalence of the disease as well as identifying carrier horses to babesiosis.