Abstract: A total of 240 serum samples collected from horses of different ages, breeds, sexes and reproductive conditions used for sporting purposes and located at main horse farms in Cairo, Egypt, were tested for Toxoplasma gondii infection. PCR and serological assays revealed that, PCR showed the higher prevalence of toxoplasmosis (53.8%) followed by LAT (52.1%), MAT (50.8%) and lowest prevalence by ELISA (39.2%). Prevalence of T. gondii infection in relation to breed, sex and reproductive condition was determined. Prevalence was higher (73.1%) in imported breeds followed by native (58.5%) and lowest prevalence in Arabian breeds (44.4%). Higher prevalence (60.8%) was detected in females represented by 10.8, 12.4 and 29.9% in fillies, pregnant and repeat breeders, respectively. On other hand, lower prevalence (23.9%) was detected in males represented by 0, 8.7 and 15.2% in colts, stallions and racers, respectively. When the data of the serological tests were compared with that of the PCR, as a reference test for toxoplasmosis, MAT had the highest sensitivity (93.8%) followed by LAT (91.5%) and the lowest sensitivity by ELISA (71.3%). On the other hand ELISA had the highest specificity (92.8%) followed by MAT (91.9%) and the lowest specificity was by LAT (88.3%). The present study is the first time to adopt PCR and serological survey of T. gondii antibodies in sport horses in Egypt and suggests that MAT alone or with LAT can be used as a highly sensitive screening test followed by PCR as a specific confirmatory test for diagnosis of toxoplasmosis in Equines. Also, the high prevalence observed indicate that the risk of infection from horses to people or other animals is very high.