The genus Atelopus is one of the most diverse of the Bufonidae family and because of the bright color, they are referred to as harlequin frogs. As they occur in mature forest areas, 97% of species are in extinction risk. One of the species, Atelopus hoogmoedi, occurs in the north and south of the Amazon River and probably represents more than one evolutionary unit. Based on this premise, our objective was to compare the individuals of A. hoogmoedi of the south and north of the Amazon river. The DNA was extracted by the phenol chloroform method from eight individuals of A. hoogmoedi, seven from Monte Alegre (north of Amazon River) and one from Anapu (south of Amazon River) and then amplified via Polymerase Chain Reaction (PCR) using the mitochondrial 16S rRNA marker. In the 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 (ABGD) in both Simple Distance and Kimura 2 Parameters models, A. hoogmoedi collected in Anapu was recognized as a distinct species from those of the Guiana Shield. Thus Atelopus 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.
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