In bitterlings (Acheilognathinae) C- and Ag-banding karyotypes of 6 species-subspecies collected in China and South Korea were analyzed. The chromosomal constitution of 2n =46 (4 SM + 42 ST) in Rhodeus atremius fangi was quite different from that of 2n = 48 (8 M + 20 SM + 20 ST) in other species-subspecies in Rhodeus. It was concluded from the analysis of banded chromosomes that the increase in number of ST during the karyotype change from 2n = 48 to 2n = 46 was achieved by a series of pericentric inversions from 24 M-SM to 24 ST, and the decrease in the diploid number was caused by an additional tandem fusion of 4 ST chromosomes, forming a new ST pair in the 2n = 46 karyotype. The karyotype of Tanakia koreensis, T. signifer, and Acheilognathus macropterus is 2n = 48 (8 M + 20 SM + 20 ST), 2n = 48 (8 M + 20 SM + 14-16 ST + 4-6 A), 2n = 44 (14M + 16 SM + 14 ST), respectively. In R. ocellatus ocellatus, T. koreensis, T. signifer and A. macropterus, karyotype changes from 2n = 48 to 2n = 44 due to centric fusion and inversion have also been estimated. It was suggested that C-banding heterochromatin was greatly concerned with the karyotype evolution in bitterlings.