Argentina is a home to millions of beef and dairy cattle and is one of the world's major exporters of meat. In the present study, Trypanosoma vivax was prevalent (2016–2018) in two major livestock farming regions, the Gran Chaco and the Pampas. In the Gran Chaco, 29% and 51% of animals (n = 72, taurine x zebuine crossbreed) were, respectively, positive by TviCATL‐PCR and the more sensitive fluorescent fragment length barcoding (FFLB) method. While 18.4/38.8% of breeding cows (n = 49) tested positive by PCR/FFLB, infection increased to 52.2/78.3% in an outbreak of acute infection in steers (n = 23, taurine breed) brought from a non‐endemic area. In the Pampas, overall infection rates in dairy cows (n = 54, taurine breed) were comparable (p > .01) between PCR (66.7%) and FFLB (62.9%) and showed a remarkable increase (PCR / FFLB) from 48.3/44.8% in 2017 to 88/84% in 2018. Infected dairy cattle exhibited anaemia, fever, anorexia, enlarged lymph nodes, emaciation and neurological signs. In contrast, beef cows (taurine x zebuine crossbreed) from the Pampas (n = 30) were asymptomatic despite exhibiting 16.7% (PCR) and 53.3% (FFLB) infection rates. Microsatellite genotyping revealed a remarkable microheterogeneity, seven genotypes in the Gran Chaco, nine in the Pampas and five shared between both regions, consistent with regular movement of T. vivax infected livestock. Data gathered in our study support the Gran Chaco being an endemic area for T. vivax, whereas the Pampas emerged as an outbreak area of acute infection in dairy cattle with critical negative impact in milk production. To the best of our knowledge, this is the first molecular study of T. vivax in Argentina, and results indicated the need for preventive measures to control T. vivax spread from the Gran Chaco to vast livestock farming areas across Argentina.