In order to investigate the cytogenetic characters of Sinibotia reevesae, the chromosome number and karyotype of S. reevesae were analyzed using a routine method including intraperitoneal injection of PHA and colchicine, nephrocyte cultivation in vivo, and slides preparation with air—drying. Erythrocyte nuclear DNA content of S. reevesae was determined by using a flow cytometer (BD Accuri TM C6) with the chicken erythrocytes (their DNA content is 2.50 pg) serving as reference cells. The results showed that S. reevesae was diploid, the chromosome number was 2n = 96, and the karyotype formula was 2n = 36m + 14sm + 20st + 26t, and arms number (NF) was 146 (Table 2 and Fig. 1). The heterosomes were not observed. The DNA content of S. reevesae was 2.60 ± 0.36 pg/2c (n = 10, Fig. 2). By comparing with the chromosome karyotypes of other 26 Cobitidae species, it was concluded that S. reevesae should belong to specialization group, and karyotype of S. reevesae might evolve from the type of Cobitidae ancestors by Robertsonian translocation of chromosomes and chromosome polyploidy. Our results could provide essential data for broodstock conservation and cytogenetic research of the S. reevesae.