To study the morphological differences between and the evolutionary mechanisms driving the differentiation of geographically distinct populations of Gymnodiptychus dybowskii, 158 fish were collected from the Turks River and the Manas River in Xinjiang from 2020 to 2021 with the approval of the Academic Ethics Committee. The morphological characteristics of the fish were assessed using classical fish ecology methods such as traditional morphometric measurements and the framework approach. The results showed that the morphological characteristics of the populations in the Turks River and Manas River were significantly different; a one-way ANOVA revealed 22 highly significant differences (p < 0.01) and 1 significant difference (p < 0.05) among the 33 morphological traits of the observed populations, and a principal component analysis revealed that there was no overlap between the two populations of G. dybowskii. The main characteristics associated with principle component 1 were the terminus of the dorsal fin to the ventral origin of the caudal fin (D—F), the dorsal origin of the caudal fin to the origin of the anal fin (E—H), and the insertion of the pectoral fin to the terminus of the pectoral fin (J—K); the main factors associated with principal component 2 were the body height (BD), the terminus of the dorsal fin to the insertion of the pelvic fin (D—I), the caudal peduncle height (CPH), and the tip of the snout to the last end of the frontal maxilla (A—B); and the main traits associated with principle component 3 were the terminus of the anal fin to the origin of the anal fin (G—H), the body width (BW), the insertion of the pelvic fin to the terminus of the pelvic (I—L), the insertion of the pectoral fin to the terminus of the pectoral fin (J—K), and the insertion of the pelvic fin to the insertion of the pectoral fin (I—J). An OPLS-DA revealed that the two populations could be wholly separated and that the intergroup growth traits of the Manas River population were different and significantly greater than those of the Turks River population. The discriminant functions of the Turks River and Manas River populations of G. dybowskii were as follows: YT = −432.033 + 1787.748X1 + 826.517X2 + 249.002X3 + 1183.050X4 + 554.934X5 + 999.296X6 + 627.428X7; YM = −569.819 + 2041.044X1 + 344.942X2 + 333.737X3 + 940.512X4 + 348.222X5 + 1167.770X6 + 1015.904X7. According to a coefficient of variation analysis, a total of nine traits, namely, EI/BL, C-D/BL, E-F/BL, F-H/BL, H-I/BL, C-J/BL, D-I/BL, D-H/BL, and D-F/BL, had a CD > 1.28, indicating that the differences in these nine traits had reached the subspecies level. The results showed that G. dybowskii significantly differed between the two geographically distinct populations in the Turks River and the Manas River and have differentiated to the subspecies level. This study provides a basis for a better investigation of the population structure of highland endemic fishes and the mechanisms by which they diverged and lays a foundation for developing and utilizing germplasm resources from endemic fishes in Xinjiang.