A Polymerase Chain Reaction (PCR) and Southern blot hybridization for the detection of Theileria annulata are described. The PCR used primers amplifying a 785 base-pair fragment of the T. annulata gene which encodes the 30 kDa major merozoite surface antigen, Tams1. The sensitivity of the PCR in bovine blood was 1 piroplasm in 1 μl of blood. T. buffeli, T. parva, Babesia bigemina, B. bovis and B. divergens were not detected. The PCR detected down to 1 infected acinus/tick in resting and partially fed adult Hyalomma anatolicum anatolicum ticks and was negative for T. lestoquardi and T. equi, which are transmitted by this tick but are not infective to cattle. The specificity of the PCR was checked using 30 stocks of T. annulata, all of which were detected. Three stocks of T. lestoquardi, 4 of T. equi and 1 each of T. buffeli, T. parva, B. bigemina, B. bovis and B. divergens were used to ascertain there were no cross-reactions. A nested PCR using separate primers for the first reaction and the same primers for the second reaction detected T. annulata to the same sensitivity and specificity in saponin-extracted DNA samples stored for long periods at −20 °C.