The genus Atelopus is one of the most diverse of the Bufonidae family; because of their bright color, they are referred to as harlequin frogs. They occur in mature tropical forest areas and in this region, these forests are under anthropic pressure and limited to fragments, which facilitates the action of pathogenic fungi. One of these toad species, Atelopus hoogmoedi, is only found to the north and south of the Amazon River. Based on genetic data this species name represents more than one evolutionary unit. To explore this premise, we compared individuals of A. hoogmoedi collected south and north of the Amazon river in the state of Pará, Brazil. The DNA was extracted by the phenol chloroform method from eight individuals, seven ©FUNPEC-RP www.funpecrp.com.br Genetics and Molecular Research 19 (1): gmr18392 G.W.B. da Silva et al. 2 from Monte Alegre (north of Amazon River) and one from Anapu (south of Amazon River) and then amplified via PCR using a mitochondrial 16S rRNA marker. Phylogenetic analysis of maximum likelihood for A. hoogmoedi revealed a paraphyletic group with three lines: French Guiana 1 and 2, Guyana and Monte Alegre, and Anapu. The genetic distance between Anapu and Monte Alegre was 2.9%. According to the Automatic Barcode Gap Discovery in both Simple Distance and Kimura 2 Parameters models, A. hoogmoedi collected in Anapu is recognized as a distinct species from those of the Guiana Shield. Thus A. hoogmoedi to the south of the Amazon River was classified as an unconfirmed candidate species, requiring more collections and access to individuals from other localities of its occurrence for confirmation.