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To explore genetic diversity and estimate the genetic differences among populations of Lutraria maxima in the coastal waters off south to southeast China, the morphology of the species of five different geographical populations (Beihai, Weizhou Island, Zhanjiang, Xiamen, and Fuzhou) in Guangxi, Guangdong, and Fujian provinces was studied statistically in combination with the microsatellite markers. As revealed by morphological principal component analysis (PCA), the cumulative contribution rate of the first three principal components was 72.596%. The discrimination accuracy ranged from 47.5% to 80.0%, and the scatter plots of principal component and discriminant analysis were consistent in overall, showing that the Xiamen and Fuzhou populations were overlapped obviously. For microsatellite markers, 10 pairs of polymorphic primers were obtained by high-throughput transcriptome sequencing, and used for genetic diversity analysis. It was showed that the average number of alleles and effective alleles observed in each population ranged from 8.100 to 10.900, and from 3.497 to 4.228, respectively. The average observed heterozygosity (Ho) and expected heterozygosity (He) in the five populations ranged from 0.541 to 0.615, and from 0.642 to 0.733, respectively. The genetic distance (DA) ranged from 0.078 to 0.523, and the population genetic differentiation index (FST) ranged from 0.027 to 0.139. The unweighted pair-population method with arithmetic means (UPGMA) and structure analysis showed that the five populations could be divided into two main clusters, the Beibu Gulf group (Beihai and Weizhou Island) and the Southeast China Sea group (Zhanjiang, Xiamen, and Fuzhou), suggesting that L. maxima has been separated geographically by the barrier of the Leizhou Peninsula into two groups in evolution, which provided us with a scientific clue to better protect the bioresource and establish an appropriate fishery management stocks for L. maxima populations in south China. [ABSTRACT FROM AUTHOR]