Characiformes is the most cytogenetically studied group of freshwater Actinopterygii, but karyotypical data of several taxa remain unknown. This is the case of Nematocharax, regarded as a monotypic genus and characterized by marked sexual dimorphism. Therefore, we provide the first cytogenetic report of allopatric populations of Nematocharax venustus based on distinct methods of chromosomal banding and fluorescence in situ hybridization (FISH) with repetitive DNA probes (18S and 5S rDNA). The karyotype macrostructure was conserved in all specimens and populations, independently on sex, since they shared a diploid number (2n) of 50 chromosomes divided into 8m+26sm+14st+2a. The heterochromatin was mainly distributed at pericentromeric regions and base-specific fluorochrome staining revealed a single pair bearing GC-rich sites, coincident with nucleolar organizer regions (NORs). On the other hand, interpopulation variation in both number and position of repetitive sequences was observed, particularly in relation to 5S rDNA. Apparently, the short life cycles and restricted dispersal of small characins, such as N. venustus, might have favored the divergence of repetitive DNA among populations, indicating that this species might encompass populations with distinct evolutionary histories, which has important implications for conservation measures.Characiformes é o grupo de Actinopterygii de água doce mais estudado citogeneticamente, porém dados cariotípicos de vários taxa permanecem desconhecidos. Este é o caso de Nematocharax, considerado um gênero monotípico e caracterizado pelo acentuado dimorfismo sexual. Em vista disso, nós fornecemos a primeira descrição citogenética de populações alopátricas de Nematocharax venustus, baseada em métodos distintos de bandamento cromossômico e hibridação fluorescente in situ (FISH) com sondas de DNA repetitivo (DNAr 18S e 5S). A macroestrutura cariotípica mostrou-se conservada em todos os espécimes e populações, independentemente do sexo, uma vez que compartilharam um número diploide (2n) de 50 cromossomos dividido em 8m+26sm+14st+2a. A heterocromatina distribuiu-se principalmente nas regiões pericentroméricas e a coloração com fluorocromos base-específicos revelou um único par portador de sítios GC-ricos, coincidentes com as regiões organizadoras de nucléolo (RONs). Por outro lado, foi observada uma variação interpopulacional no número e na posição das sequências repetitivas, especialmente em relação ao DNAr 5S. Aparentemente, ciclos de vida curtos e dispersão restrita dos pequenos caracídeos, tal como N. venustus, podem ter favorecido a divergência do DNA repetitivo entre as populações, indicando que essa espécie pode englobar populações com distintas histórias evolutivas, o que tem implicações importantes para medidas de conservação.

An integrative approach based on morphological and multilocus genetic data was used to describe a new species of Nematocharax from the headwaters of the upper Contas River on the Diamantina Plateau, north‐eastern Brazil and to infer the relationships among evolutionary lineages within this fish genus. Multispecies coalescent inference using three mitochondrial and five nuclear loci strongly supports a basal split between Nematocharax venustus and the new species, whose distinctive morphological characters include absence of filamentous rays on pelvic fins of maturing and mature males, reduced anal‐fin lobe length and lower body depth. The unique morphological and genetic traits of the population from the upper Contas River were supported by previous reports based on cytogenetics, DNA barcode and geometric morphometrics, reinforcing the validation of the new species. The conservation status of this new species is discussed.