Abstract: The morphometrical variability of six natural populations of Difflugia tuberspinifera was investigated using the statistic methods based on 374 samples from Yangtze River and Pearl River valleys. The size frequency distribution analysis indicated that D. tuberspinifera is a size-monomorphic species with normal distribution of shell height, shell diameter and aperture diameter. The size of spine length, collar height, foreside length and number of conical spines are the most variable. The correlation analysis showed that most characters are inter-correlated with P＜0.05. The shell height, shell diameter, aperture diameter, spine length, collar height, rear end length, foreside length, number of aperture tooth-like structures and number of conical spines differed significantly between different populations (P＜0.0001), but principal component analysis (PCA) did not clearly distinguish the six populations based on the morphometric data. However, the cluster analysis separated the six populations into two groups, the Pearl River-Yangtze River group and the Yangtze River group. Further, the populations with different shell characters can coexist within the same river valley. Thus, there is a high morphological variability or diversity within the populations of D. tuberspinifera which is related with local environmental conditions.