To explore the morphological differentiation of Schizothorax o’connori populations in the Yarlung Tsanpo River, multivariate morphometric methods were used to investigate the morphological differences of ten populations of S. o’connori based on the proportional data of 12 conventional characters and 20 truss characters. In principal component analysis, six principal components (PC) were constructed and their cumulative contribution rate was 74.23%, of which the first principal component (PC1) was mainly affected by 14 characters along horizontal body axis such as TL/BL, SL/ BL, ED/ BL, D1-2/ BL and D1 -3/BL, and the PC2 is mainly affected by 10 characters along vertical body axis such as BD/ BL, CPD/ BL, BW/BL, DOP/BL, DOV/BL. The results of principal component analysis, one-way ANOVA and cluster analysis showed that there was distinct morphological differentiation between the two populations (Tongmai and Kadaqiao) from the Purlung Tsangpo River - a tributary in the lower reaches of the Yarlung Tsangpo Grand Canyon (YTGC), and the eight populations from the upper reaches of the YTGC, and each of the two groups was clustered into one branch. Whereas the eight populations from the upper reaches of the YTGC were grouped into two sub-groups. The discriminant functions were built for ten populations of this species, with a total discriminant accuracy rate of 88.60%, of which the discriminating accuracy for Shigatse, Lhatse, Maldrogongkar and Tongmai populations ranged from 95.92 % to 100.00%. The morphological differentiation between the S. o’connori populations from the upper and lower reaches of the the YTGC may be related to geographical isolation and their adaptation to specific habitats. The results obtained in this study will provide basic information for the identification, enhancement and release, conservation of germplasm resources, and adaptive evolution study of this species.