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7 Pág., The family Trichechidae (order Sirenia) comprises three species African (Trichechus senegalenses), West Indian (T. manatus), WIM)], and the Amazonian manatees (T. inunguis, AMM). Whereas WIM inhabits both riverine and coastal systems in the western Atlantic, AMM is the only exclusively freshwater sirenian, endemic to the Amazon River Basin. The study of infectious agents is essential to species conservation, especially considering that both species are classified as Vulnerable by the IUCN Red List and as Endangered by the Brazilian Red List. The current knowledge about viral agents in sirenians is scarce. Herpesviruses and adenovirus are DNA viruses able to infect and cause disease in a wide range of hosts. Herein, we used panPCR protocols to survey herpesvirus and adenovirus in blood samples of wild WIM (n = 23) and AMM (n = 26) under human care in Brazil. Herpesvirus DNA was detected in one juvenile female WIM (1/23; 4.3%; 95% CI -4.7 – 13.3) from Ceará state and in four AMM (two juvenile females, a juvenile male, and an adult female; 4/26; 15.4%; 95% CI 0.5 – 30.3) from Amazonas state. The two different gammaherpesvirus DNA polymerase sequence types identified (one per species, a sequence type in a WIM and another one in three AMM) were highly similar (99% nucleotide identity) to Trichechid herpesvirus 1, reported in West Indian manatees of Florida (USA), and 100% identical when translated into amino acids. A herpesviral glycoprotein B sequence was identified in two AMM. None of the samples was positive to adenovirus. To the authors’ knowledge, this is the first herpesvirus detection in manatees from South America, expanding the herpesvirus geographical range, and the first in WIM and AMM worldwide. Our findings suggest (i) that West Indian and Amazonian manatees are possibly the natural hosts of the detected herpesvirus, and (ii) coevolution of that gammaherpesvirus with Trichechus. Future studies are necessary to characterize the obtained virus and elucidate potential pathological effects (if any) in these species., We thank the staff of AQUASIS and INPA for their logistical and technical support. We thank the Sao Paulo Research Foundation (FAPESP), the Brazilian National Council for Scientific and Technological Development (CNPq), and the Coordination for the Improvement of Higher Education Personnel (CAPES) for their support. Ana Carolina Ewbank and Samira Costa-Silva receive PhD Grants from FAPESP (process numbers 2018/20956–0 and 2020/12434–9). Aricia Benve nuto and Roberta Zamana Ramblas receive PhD Grants from CNPq [process numbers 141868/2019–8 and 165364/2018–1]. Carlos Sacristan and Irene Sacristan receive Juan the la Cierva incorporacion fellowships under process numbers IJC2020–046019-I and FJC2020–046311–1, respectively. Jose Luiz Catao-Dias is the recipient of a professorship from CNPq (304999–18). This study was approved by the Ethical Committee in Animal Research of the School of Veterinary Medicine and Animal Sciences, University of Sao Paulo (Process number 7151291019). The samples used in this study were collected in full compliance with specific federal permits issued by the Brazilian Ministry of Environment and approved by the Biodiversity Information and A.C. Ewbank et al.