Understanding the adaptive responses of fish to different local habitats is important for assessing fragmentation effects and directing conservation management. In this study, morphological and genetic differentiations were tested among Hemiculter leucisculus populations from three lakes, i.e., the Liangzi, Niushan, and Biandantang Lakes, fragmented from the Liangzi Lake complex in the middle Yangtze. These lakes have different habitat environments, largely influenced by their size and connection patterns with the mainstem, and also have different timings of fragmentation in the complex. Morphological differentiations among populations in the lakes were expected to reflect population responses to local habitat conditions. Genetic differentiation was expected to occur due to isolation of the populations. Our results revealed statistically significant morphological differences and no genetic differentiation among populations from the lakes. Morphometric differences might be related to swimming ability of the fish in different habitats. Population genetics results showed that all individuals constituted a single population. However, a recent reduction in gene flow between the lakes, a loss of alleles, and low genetic diversity were found, suggesting probable genetic impacts of fragmentation. These results suggest that phenotypic plasticity might contribute to morphological differences in the differing local conditions, and this interpretation needs confirmation from further experiments testing the roles of these morphological differences in swimming ability and the differing environments in generating these differences. Our results have led to a better understanding of the responses of fish populations to habitat fragmentation and can thus direct the connectivity restoration of fragmented habitats in the Yangtze River.