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Trypanosoma vivax is a protozoan parasite that causes trypanosomosis in ruminants and is widely distributed in tropical areas in the world. The control of this disease depends on the sensitivity and specificity of the diagnostic tests implemented for naturally infected samples, where parasitaemias are usually low. This study aimed to evaluate the analytical sensitivity and specificity of several primers for T. vivax detection in experimental infections and their implementation for the diagnosis of trypanosomosis in naturally infected bovine and ovine samples. Using a T. vivax Venezuelan isolate, five sets of primers were evaluated: TviSL1/2, ITS1CF/BR, TVMF/R, ILO1264/1265, TVWA/B. Additionally, we tested the PCR protocols using different DNA quantities. The best set of primers (ILO1264/1265) was used to detect T. vivax DNA from whole blood and buffy coat samples of 12 sheep (ovine) and 45 cattle (bovine) of small farms from Venezuela, and compared to the micro-haematocrite centrifugation technique (MHCT). The highest sensitivity was 0.0001 ng for ILO1264/1265 and TVWA/B primers. Using 100 ng of DNA extracted from the buffy coat and the ILO1264/1265 primers for trypanosomosis diagnosis from naturally infected samples, yielded 66.7% (8/12) and 35.7% (16/45) positives in ovine and bovine respectively. The percentage of positives samples increased to 83.3% (10/12) and 64.4% (29/45), with 300 ng in the assays. Contrary, using 300 ng of DNA extracted from the whole blood yielded only 50% (6/12) and 28.9% (13/45) of positives samples for T. vivax respectively. MHCT only detected the parasite in bovine samples with 17.8% (8/45) of positives. Based on our results, we recommend the use of the ILO1264/1265 primers and 300 ng of DNA extracted from the buffy coat for epidemiological studies of naturally infected animals. Moreover, detection of the parasite in ovine herds highlights a possible role of this host in the epidemiology of trypanosomosis in Venezuela., (Copyright © 2021 Elsevier B.V. All rights reserved.)