We describe a new extant species of freshwater turtle from Brazil: Mesoclemmys sp. nov., a medium-sized toad-headed turtle (mean carapace length = 189.5 ± 25.8 mm), which to date has only been found in the Brazilian Amazon Basin, western Pará State, Central Amazon, and the lower Amazonas River Basin. It is easily distinguished from its sympatric congeners by differences in body size, head size, carapace shape, and coloration. The head is triangular and completely black, with large red eyes positioned anteriorly and a pair of long, light yellow barbels positioned anteriorly close the mandible. The carapace is oval and colored dark reddish brown. The plastron is black in the central region and burnt-yellow along the edges and ventral aspects of the marginal scutes and bridge, with dark seams. The plastral formula is 3-5-7-4-1-6-2. The new species inhabits temporary rainwater ponds under closed-canopy rainforest and has a known geographic range of 2183 km2. Sequences of fragment mitochondrial DNA (cytochrome c oxidase subunit I and 16S) of the new species were determined and compared with the available GenBank sequences. Our phylogenetic results indicate that the specimens represent a divergent taxon within Mesoclemmys and a robust analysis of morphological data corroborates the existence of a new distinct species of Mesoclemmys, described herein.