Amazophrynella is a genus of the family Bufonidae, currently represented by three species. The type species of the genus, Amazophrynella minuta, however, is a complex of species occurring throughout the Amazonian biome. This group remains problematic taxonomically; the difficulty lays principally in the lack of diagnostic characters in the original description of A. minuta, the lack of molecular data and refined taxonomic comparison of individuals through its wide distribution. We describe a new species of the genus Amazophrynella, distributed in the southwestern part of the Guiana Shield of Brazil based on a series of morphological and molecular characters. The new species differs from others of the genus by presenting a slightly truncated triangular snout, ventral texture covered by many fine granules, white belly covered with black spots and by fifteen molecular autapomorphies in the 16S rDNA fragment. Uncorrected p-distances of a fragment of the 16S mitochondrial rDNA gene revealed high divergence among other Amazophrynella species (9%-14%). Additionally we provide a new diagnosis of topotypic material of Amazophrynella minuta including molecular data. Our results show the existence of a subestimated diversity in the genus Amazophrynella.