Abstract: Fishway is an important channel for migration and gene exchange of fish populations between upstream and downstream of dam. In this study, we sequenced 220 Cyt b gene sequences of Schizothorax waltoni, and analyzed the genetic diversity and genetic structure from five populations, including upstream of Zangmu Hydropower Station (ZMBS), Zangmu fishway (ZMYD), downstream of Zangmu Hydropower Station (ZMBX), Jiacha fishway (JCYD) and downstream of Jiacha Hydropower Station (JCBX). Our aims were to study the need for assisted gene flow to prevent further loss of genetic diversity and boost adaptive potential and genetic selectivity of fishways on S. waltoni. The results show that: (1) The level of genetic diversity for S. waltoni populations between upstream and downstream of Zangmu Hydropower Station and Jiacha hydropower station (ZMBS, ZMBX and JCBX) were similar (Hd (ZMBS)=0.868, Pi (ZMBS)= 0.004 16; Hd (ZMBX)=0.825, Pi (ZMBX)=0.003 88; Hd (JCBX)=0.833, Pi (JCBX)=0.003 70), gene flow was not blocked (25.23＜|Nm|＜81.33), genetic divergence was not detected (-0.020 2＜FST＜0.006 1；P>0.05), indicated low risk of outbreeding depression. Five geographic populations were defined as one genetic management unit. There exists the demand of gene flow to pass through Zangmu fishway and Jiacha fishway for S. waltoni to avoid inbreeding depression. S. waltoni should be a target fish for Zangmu fishway and Jiacha fishway; (2) The results of network and neighbor-joining phylogenetic tree showed three Clades of S. waltoni, indicated sympatric divergence but not cryptic species, which may be formed by the secondary contact after the Kunlun-Yellow River Movement or the Pleistocene glacial refugia. (3) Genetic selection pressure was detected on S. waltoni from Zangmu fishway and Jiacha fishway. The results could provide data support and theoretical implications for rechecking the rationality of target fish setting and promoting the refinement operation management of fishway.