Chiao-Chuan Han, Tsair-Bor Yen, Nian-Cih Chen, and Mei-Chen Tseng (2017) Both Onychostoma barbatulum and O. alticorpus are primary freshwater fish in Taiwan. The former has been developed as an aquaculture species with high economic value, while the latter is a native endemic species in Taiwan. Understanding the cytogenetic information of these two species is necessary for their selected breeding, recovery, and management. In this study, Giemsa staining, silver-binding nucleolar organizer region (Ag-NOR), C-banding, and fluorescence in situ hybridization (FISH) with 18S ribosomal (r)DNA probes were used to analyze the cytogenetic characteristics. Results of Giemsa staining showed that the two Onychostoma species shared the same number of chromosomes, 2n = 50. Respective karyotype formulas of the female and male were 10 m + 22 sm + 10 st + 8 t and 11 m + 22 sm + 10 st + 7 t in O. barbatulum, and 14 m + 18 sm + 8 st + 10 t and 15 m + 18 sm + 8 st + 9 t in O. alticorpus. Karyotypes of both species showed a pair of heteromorphic chromosomes in male fish. Their sex determination should be the XX/XY system. Two pairs of Ag-NORs were found in O. barbatulum, but only one pair occurred in O. alticorpus. C-banding areas were observed on centromeres or telomeres of some chromosomes. FISH revealed different cytogenetic characters between these two species. The above cytogenetic information will contribute to species identification, population recovery, and advantages for breeding and management in the future.