Trypanosoma evansi is a widespread and neglected zoonotic parasite that afects domestic and wild animals, causing a disease commonly known as “surra.” The Brazilian Pantanal wetland is recognized as an enzootic area for this protozoan, yet recognizing the importance of reservoir hosts also in order to prevent zoonotic outbreaks. This study aimed to assess the occurrence of T. evansi in jaguars (Panthera onca) from the Brazilian Pantanal wetland and explore associated clinical and hematological manifestations. A total of 42 animals were screened by PCR and sequenced for species identifcation when positive. Trypanosoma evansi was detected in six free-ranging jaguars (six positive animals of 42 captures and 16 recaptures), representing the frst molecular evidence of such infection in this animal species. Our fndings suggest that jaguars may act as reservoir hosts of T. evansi in the Brazilian Pantanal wetland. The better understanding of the role of wildlife in the epidemiology of T. evansi is also of importance to future reintroduction and translocation programs toward wildlife conservation eforts.