In the present study, 37 buffalo from different localities in Upper Egypt were sampled and examined for Theileria spp. infection using Giemsa blood smears, conventional and nested polymerase chain reaction. The analysis targeted the 18S srDNA gene followed by sequencing and phylogenetic analysis using neighbor-joining (NJ) method. The study came up with the conclusion that Egyptian buffalos (Bubalus bubalis) infected with three Theileria (T. uilenbergi (13.5%), T. lestoquardi (2.7%) and T. ovis (2.7%)) small ruminants species rather than Theileria annulata (32.4%).