This paper reports the first account about dynamic changes on genetic diversity and population structure of Leptobotia microphthalma in the Yangtze River drainage due to dam constructions. The genetic diversity and population structure of twelve populations of L. microphthalma collected in 2010 and 2020 were estimated using 12 nuclear microsatellite markers. Reduction of genetic diversity between 2010 and 2020 was not significant in a paired t-test (p > 0.05), but population structure of L. microphthalma had a tendency to change: the genetic differentiation (Fst) among the five populations collected in 2010 were all insignificant (p > 0.05). However, differentiation (Fst) among some populations collected in 2020 were significant (p < 0.05), which indicated the population structure of L. microphthalma was changing. Correlation analysis indicated that negative correlations between the genetic diversities and geographical elevations among populations were significant for seven populations collected in 2020 (r = -0.819, p = 0.039), which means that populations of L. microphthalma in high elevation regions were more vulnerable than those in low elevation regions. Finally, some suggestions for conservation and restoration are proposed, such as artificial propagation, to prevent the further reduction of genetic diversity and population resources.