Anaplasma phagocytophilum is the causative agent of equine granulocytic anaplasmosis (EGA). This study aimed to perform serological and molecular surveys of A. phagocytophilum in thoroughbred horses from racecourses in Chile. Additionally, hematological findings related to A. phagocytophilum molecular positivity were addressed, and phylogenetic analysis of selected positive samples was performed. Complete blood count and msp2 gene real-time PCR were performed in 457 thoroughbred horses from three racecourses located in three different cities of Chile (Santiago, Viña del Mar and Concepción). Sera from horses in two racecourses (Santiago and Vina del Mar) were tested by Indirect fluorescent antibody test (IFAT) to detect IgG antibodies against A. phagocytophilum. The occurrence of A. phagocytophilum by real-time PCR was 13.6 % (62/457, 95 % CI: 10.8–16.3 %), with the highest occurrence observed in Santiago (26.5 %), followed by Concepción (9%), and the lowest in Viña del Mar (5%). The overall frequency of IgG antibodies to A. phagocytophilum was 7.9 % (23/290, 95 % CI: 4.8–12.7 %), with 9.9 % in Santiago and 6.5 % in Viña del Mar. Only three animals from Santiago Racecourse were positive in both real-time PCR and serology. PCR-positive horses from Santiago racecourse presented significantly lower hemoglobin, mean corpuscular value (MCV), and mean corpuscular hemoglobin concentration (CHCM), and higher eosinophil counts. Phylogenetic analysis based on the msp2 gene showed that A. phagocytophilum sequences found in the present study were closely related with A. phagocytophilum sequences from the USA and Europe. Anaplasma phagocytophilum DNA is detected for the first time in Chile.