The cyprinids, freshwater perciform fish belong to the subfamily Cyprininae. Among them, the genus Osteochilus contains 11 recognized valid species. Here, karyotype and chromosomal characteristics of Hypsibarbus malcolmi and H.wetmorei were examined applying conventional and nucleolar organizing region (NORs) staining with molecular cytogenetics.The diploid chromosome number (2n) of H.malcolmi was 50, fundamental number (NF) 62, and the karyotype displayed 8m +4sm +38 a with NORs being located at a centromeric and telomeric position of the short arms of chromosome pairs 1 and 2 ,respectively. 2n of H.wetmorei was 50, NF 67, karyotype 14m +14sm +22 a with the NORs at the telomeric position of the short arm of chromosome pairs 2. 2n and NF in males and females fish 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 chromosome. Besides, the (GAA)n microsatellites were distributed along all chromosomes of H. malcolmi, but a strong hybridization pattern in the centromeric region of a single pair in H. malcolmi, but gave a strong hybridization pattern in the centromeric region of a single pair in H. wetmorei. These cytogenomic different patterns across the genomes of these Hypsibarbus species are markers for specific evolutionary differentiation inside these two species.