DNA barcoding represents a reliable and powerful way to discriminate and identify species using a standardized region of the mt COI gene. However, a correct identification requires two factors: differentiation and assignment. When one component is lacking, the barcode approach usually fails. To circumvent such problem, we developed a dual approach using a nuclear marker as complementary identifier. A first step consisted in characterizing the first intron of the F-RTN4 gene. This intron was found to be the longest, the most divergent and the most variable of the different introns constituting F-RTN4, making it a candidate of choice. This dual approach was applied to a group of closely related armoured catfishes constituting the Panaque group within the Guianas. Three groups were found: Pseudacanthicus, Hemiancistrus, and Peckoltia-Panaqolus, and four new species were highlighted. Within the latter group, Panaqolus koko n. sp. displayed a pattern of mitochondrial introgression with Peckoltia otali n. sp., while Peckoltia capitulata n. sp. and Peckoltia simulata n. sp. revealed cryptic species of Peckoltia oligospila. Hemiancistrus appeared significantly distinct from Peckoltia. Its type species is redescribed and a neotype is designated to clarify its taxonomic status considering the loss of the holotype.