Parasitic nematodes within Onchocercidae are a diverse group transmitted by hematophagous arthropods. This study investigated the molecular occurrence of filarioid nematodes in 93 wild mammals from the Amazon, Cerrado and Pantanal biomes in Brazil, based on the analysis of the mitochondrial genes 12S ribosomal DNA gene (12S rDNA) and cytochrome c oxidase subunit I gene (COI). Conventional polymerase chain reaction (cPCR) targeting the 12S rDNA gene yielded positive results in 14·44% (13/93) of the samples, including 9·86% of jaguars (7/71), 50% of pumas (1/2), 12·5% of giant anteaters (1/8), 50% of ocelots (1/2) and 60% of crab-eating foxes (3/5). Among the 12S-positive samples, 46% (6/13) also tested positive for the COI gene; however, only 1 sequence was suitable for further analysis. Phylogenetic analyses based on 12S gene sequences revealed 4 distinct lineages within the family Onchocercidae. Groups Ia and Ib, composed of Cerrado and Pantanal sequences from jaguars, formed sister clades to Brugia pahangi and Malayfilaria sofiani, respectively. The sequence from the giant anteater (Group Ic) was more divergent, forming a sister clade to species of the genera Malayfilaria, Wuchereria, and Brugia. Group II included sequences closely related to Dirofilaria immitis and D. striata, encompassing samples from crab-eating foxes, ocelots and a puma. These findings suggest that several wild mammal species may serve as reservoirs for previously uncharacterized Onchocercidae nematodes. Our findings expand the existing knowledge on host associations of filarioid nematodes infecting wild mammals from the Pantanal, the Cerrado and the Amazon Rainforest.