Background and Aim:The prevalence of surra in domestic cat is seldom and it is caused byTrypanosoma bruceiandTrypanosoma evansi. However, molecular diagnostic approaches are required owing to similarities in their morphology. In Yogyakarta, a domestic cat was diagnosed with trypanosomiasis; however, the causative species was undetermined. Therefore, we aimed to molecularly and biologically identify the isolate.Materials and Methods:Approximately 1 mL of blood from an infected cat was collected into EDTA tube and separated for inoculation into donor mice, blood smear, and DNA isolation. Two donor mice was then used for increasing the number of parasite in order to infect 10 experimental mice. Parasitemia was monitored daily in each experimental mouse by preparing a wet mount and Giemsa-stained thin blood smear. The blood of experimental mice that reached the peak of parasitemia was then collected and used for DNA isolation. Each blood sample, which collected from infected cat and experimental mice, was then isolated and amplified the DNA by polymerase chain reaction using ITS-1. The parasitemia pattern and viability of the animals were observed to determine the biological characteristics of trypanosomatid, while to assess the molecular characteristics, the internal transcribed spacer (ITS)-1 amplification was used.Results:The prepatent period of this trypanosomatid is between 2 and 4 dpi, whereas the life span of mice is approximately 4–10 dpi. Morphologically, the trypomastigote in the cat blood smear had long slender and intermediate shapes. However, only the long slender form was detected. Among the total of 410 nucleotides (NT) of ITS-1 sequences, 25 NT substitutions differed between the cat and mouse isolates. Phylogenetic analysis revealed that both samples had a close genetic relationship withT. evansi.Conclusion:Trypanosoma evansi, a highly virulent trypanosomatid, was isolated from a cat in Yogyakarta.