Hemotropic Mycoplasma spp., also known as Hemoplasmas, infect several species of domestic and wild animals, including humans. Although infections in wild animals are mostly asymptomatic and chronic, there is a risk of spreading the patogen to other susceptible animals. They can cause death in immunosuppressed mammals, and have implications for the conservation of already threatened wild mammals. The objective of this study was to report on the molecular detection of Mycoplasma spp. hemotropic. in wild mammals run over in the Southern Brazil, describing new findings of hemoplasmas in this region. Carcasses of 24 road-killed wild animals were collected on the highways of cities belonging to the Pelotas microregion, in the south of RS, Brazil, and were necropsied. Ectoparasites were removed and classified according to morphological taxonomic keys. Blood samples from all animals were collected and genomic DNA was extracted. Polymerase chain reaction (PCR) amplification targeting the 16S rDNA and 23S rRNA genes revealed the presence of Mycoplasma spp. DNA in blood samples from 87.5% of animals. This study reports the first molecular finding of hemotropic mycoplasma in Brazilian guinea pig (Cavia aperea) and pampas fox (Lycalopex gymnocercus). The detection of hemoplasma in different host species demonstrates the spread of the agent among wild mammals in southern Brazil.