To explore differences in the Coilia mystus populations of the Yangtze Estuary and its adjacent waters, 240 individuals were collected from four populations of Lyusi (Ls population), Chongming Island in Shanghai (Cm population), Zhoushan (Zs population) and Wenzhou (Wz population). Twenty-one morphological parameters of the sagittal otolith were measured by otolith measurements and the otolith frame method. Twenty morphological indices transformed from the 21 morphological parameters were analyzed by multivariate statistical analyses. Non-parametric tests showed that there were significant differences in 14 of the morphological indices among the 4 populations. Principal component analysis constructed 7 principal components of the otolith morphological characteristics, with a cumulative contribution rate of 81.79%. The overall shape of the otoliths was similar among populations, and the differences were mainly reflected in the local frame morphological indices. The discriminant equation of the 4 populations was constructed using the 9 parameters with the greatest contribution rates. The discriminant accuracy of the Wz population was highest (96.7%), followed by the Cm (66.7%), Ls (60.0%), and Zs populations (58.3%). Cluster analysis showed that the Cm and Zs populations were closest, followed by the Ls population, and the Wz population had the furthest distance. The shape of the sagittal otolith in the Cm population was similar to the Zs and Ls populations, and there were significant differences in the otolith morphology of the Wz population versus the other 3 populations. These results indicate that Wz is an ecological population. Therefore, the differences in otolith morphology between populations of C. mystus with short-distance migratory habits are related to their geographical distribution. Otolith shape varied little among geographically close groups with similar habitats but was larger in groups with geographic barriers and genetic differentiation. Variability in the size of the otolith is related to the degree of geographic isolation.