The study investigated the occurrence of trypanosomosis in water buffaloes using parasitological and molecular methods. Buffaloes of different ages and both sexes comprising Bulgarian Murrah and some crosses were used in the study. Two of the 145 samples (0.13%) were positive for Trypanosoma evansi in both, blood parasite examination and PCR. One female buffalo (1/54 samples; 1.86%) from a lactating herd in the city of Muñoz, Nueva Ecija (Region 3) and another (1/51 samples; 1.96%) non-pregnant female belonging to caraheifer population from Los Baños, Laguna (Region 4) were found infected by T. evansi. The positive animals were in the age group of 3-4 years. Blood samples obtained from 40 buffaloes from Cagayan (Region 2) were found negative for trypanosome infection.