This study was carried out to identify the Trypanosoma species in symptomatic and asymptomatic horses and donkeys in Egypt by using polymerase chain reaction (PCR). Blood samples were collected from 80 horses and 37 donkeys from different farms and private owners at Cairo and Giza governorates. Thin blood smears were prepared from each sample and examined microscopically. Four samples for Trypanosoma species were positive by using microscopic examination of stained blood smears from horses. While, all samples collected from donkeys were negative for Trypanosoma species infection. Microscopic examination of stained thin blood smears revealed that the parasite were monomorphic thin trypomastigote parasite, long free flagellum and thin posterior extremity with subterminal small kinetoplast and length ranges from 18-34 micron (mean 26). The four positive samples from horses were subjected to PCR using three sets of Trypanosoma species specific primers (T. brucei group, T. evansi and T. equiperdium). Three of the 4 positive samples showed symptoms of Trypanosoma infection and one case asymptomatic. PCR assay. identified that the 4 samples were positive for T. brucei group and Trypanosoma evansi species using Trypanosoma brucei TBR 1/TBR 2 specific primer at 164 bp and T. evansi EVA 1 /2 specific primer at 138 bp While, the same four samples showed negative results using T. equiperdium Nade 5 specific primer at 395 bp. Six negative samples from horses and donkeys examined microscopically were subjected to PCR examination. These samples were negative for the three species of Trypanosoma. From this it can be concluded that, PCR assay was a highly sensitive diagnostic technique, for exact discrimination between the species of Trypanosoma infection (T. brucei group, T. evansi and T. equiperdium) in equines. Therefore, Trypanosoma infection in horses in Egypt is most probably due to T. evansi.