Equine piroplasmosis (EP) is a reportable disease according to the World Organization for Animal Health (WOAH), caused by Babesia caballi, Theileria equi and T. haneyi. This syndrome is prevalent in tropical and subtropical regions, including various areas in Brazilian. This study examines EP in the Distrito Federal, Brazil, focusing on prevalence, epidemiological characteristics, and circulating genotypes. Epidemiological data and whole blood samples were collected from horses in the Distrito Federal. DNA was analyzed using qPCR for Theileria sp. and B. caballi, followed by cPCR for 18S rDNA of Theileria sp and rap-1 gene for B. caballi and phylogenetic analysis. Results showed a high EP prevalence (56.23%) among the horses: 49.88% for Theileria sp, 1.47% for B. caballi and 4.89% for both. Phylogenetic analysis indicated genetic similarities with other countries, identifying of T. equi genotypes A and C and B. caballi genotype A. Factors linked to infection, included animals older than five years (p-value = 0.04
OR = 1.71, CI = 1.01-2.87) and health conditions (p-value = 0.049
OR = 1.91, CI = 1.003-3.64) as significantly associated with positivity for Theileria sp. and/or B. caballi. This study contributes to the awareness of the circulation and distribution of T. equi and B. caballi genotypes in Brazil, highlighting the importance of health condition and age as associated factors for infection. These findings deepen our understanding of the need for ongoing surveillance and control measures to manage EP effectively.