Cyprininae are a highly diversified but demonstrably monophyletic lineage of cypriniform fishes. Here, the karyotype and chromosomal characteristics of Hypsibarbus malcolmi (Smith, 1945) and H. wetmorei (Smith, 1931) were examined using conventional, nucleolus organizing regions (NORs) and molecular cytogenetic protocols. The diploid chromosome number (2n) of H. malcolmi was 50, the fundamental number (FN) was equal to 62, and the karyotype displayed 8m + 4sm + 38a with NORs located at the centromeric and telomeric positions of the short arms of chromosome pairs 1 and 2, respectively. 2n of H. wetmorei was 50, FN 78, karyotype 14m + 14sm + 22a with the NORs at the telomeric position of the short arm of chromosome pair 2. 2n and FN in males and females were identical. Fluorescence in situ hybridization using different microsatellite motifs as probes also showed substantial genomic divergence between both studied species. In H. wetmorei, (CAG)n and (CAC)n microsatellites accumulated in the telomeric regions of all chromosomes, while in H. malcolmi, they had scattered signals on all chromosomes. Besides, the (GAA)n microsatellites were distributed along all chromosomes of H. malcolmi, but there was a strong hybridization pattern in the centromeric region of a single pair in H. wetmorei. These cytogenomic difference across the genomes of these Hypsibarbus Rainboth, 1996 species are markers for specific evolutionary differentiation within these two species.