With an estimate of around 9,000 species, the Neotropical region hosts the greatest diversity of freshwater fishes of the world. Genetic surveys have the potential to unravel isolated and unique lineages and may result in the identification of undescribed species, accelerating the cataloguing of extant biodiversity. In this paper, molecular diversity within the valuable and widespread Neotropical genus Hoplias was assessed by means of DNA Barcoding. The geographic coverage spanned 40 degrees of latitude from French Guiana to Argentina. Our analyses revealed 22 mitochondrial lineages fully supported by means of Barcode Index Number, Automatic Barcode Gap Discovery and phylogenetic analyses. This mtDNA survey revealed the existence of 15 fully supported mitochondrial lineages within the once considered to be the continentally distributed H. malabaricus. Only four of them are currently described as valid species however, leaving 11 mitochondrial lineages currently “masked” within this species complex. Mean genetic divergence was 13.1%. Barcoding gap analysis discriminated 20 out of the 22 lineages tested. Phylogenetic analyses showed that all taxonomically recognized species form monophyletic groups. Hoplias malabaricus sensu stricto clustered within a large clade, excluding the representatives of the La Plata River Basin. In the H. lacerdae group, all species but H. curupira showed a cohesive match between taxonomic and molecular identification. Two different genetic lineages were recovered for H. aimara. Given the unexpected hidden mitochondrial diversity within H. malabaricus, the COI sequence composition of specimens from Suriname (the type locality), identified as H. malabaricus sensu stricto, is of major importance.

Austrolebias wichi, new species, is herein described from seasonal ponds of the Bermejo river basin in the Western Chacoan district in northwestern Argentina. This species was found in a single pond, a paleochannel of the Bermejo River, which is seriously disturbed by soybean plantations surrounding it. Despite intensive sampling in the area, this species was only registered in this pond where it was relatively scarce. Therefore, we consider this species as critically endangered. This species is the sister species of A. patriciae in our phylogenetic analyses and is similar, in a general external aspect, to A. varzeae and A. carvalhoi. It can be distinguished among the species of Austrolebias by its unique color pattern in males. Additionally, from A. varzeae by presenting a supraorbital band equal or longer than the infraorbital band (vs. shorter) and from A. patriciae by the convex dorsal profile of head (vs. concave). Further diagnostic characters and additional comments on its ecology and reproduction are provided.

Specimens of Imparfinis were recently collected in north‐western Argentina from the Bermejo River basin (Salta and Jujuy Provinces), del Valle River (Salta Province) and Horcones River (Santiago del Estero Province). An integrative approach to taxonomy, combining a detailed morphological study and molecular phylogenetic analyses, was applied to determine the species identity of these specimens. A principal components analysis of morphological data clustered the specimens from north‐western Argentina and from the Amazon basin, indicating a close morphological resemblance. Also, a molecular phylogenetic analysis showed populations of I. guttatus from Argentina and Peru forming a clade. According to the conducted haplotype network analysis these populations are distinct in two mutations. Thus, in the absence of morphological or molecular data indicating the contrary, the combined method supports the identity of the specimens from the tributaries of the Paraguay River in Argentina as I. guttatus, whose type locality is in the upper Beni River basin in Bolivia. This contribution is also the first record for this species from Argentina. The disjunct distribution of I. guttatus provides new evidence reinforcing the hypothesis for the origin of the Paraguayan ichthyofauna. We also provide an approach to the phylogenetic relationships of Imparfinis in Heptapteridae.