Litopenaeus vannamei is a major cultured shrimp, accounting for 70% of the total output of mariculture shrimp in China. Genetic improvement is an essential measure for improving the important economic traits in shrimp aquaculture. Understanding the genetic diversity and population structure of different populations using molecular markers is considered as a prerequisite for efficient germplasm exploitation and utilization to deploy suitable breeding program for genetic improvement. Using the criteria of motif repeats, a total of 10 453 975 potential SSRs with a minimum of five repetitions were identified from the 1 663 588 777 bp genome sequence by Perl script of MISA, which accounts for 18.56% of the total length of the genome sequence with a distribution density of 6 589.25 loci/Mb. A total of 42 SSR primer pairs were randomly selected for validation, and 14 SSR loci had allelic polymorphism. The genetic characteristics of three representative domestic populations including Guihai No.1 (CG), Haixingnong No.2 (CH) and Shandong population, and three foreign populations including Ecuador No. 1 (FO), Ecuador No. 2 (FT) and Mexico (FM) were evaluated using 14 polymorphic microsatellite markers. The results showed that 208 alleles were detected in six populations, the average allele number, average expected heterozygosity, average observed heterozygosity and polymorphism information content were 14.9, 0.462, 0.736 and 0.7411, respectively. The genetic diversity of six populations ranged from high to low: CH > FO > FM > CT > FT > CG. There was medium genetic differentiation between the Guihai No.1 and three foreign populations. The analysis showed that Guihai No.1 and Haixingnong No.2 clustered into one branch, while Shandong population and three foreign populations including Ecuador No. 1, Ecuador No. 2 and Mexico clustered into another branch. In conclusion, the 14 polymorphic markers could be used as efficient markers to analyze the genetic diversity of L. vannamei population, and the furthest distance was found between Guihai No. 1 and three foreign populations. This study analyzed the genetic background of six important populations of L. vannamei, which is of great significance to its development of breeding program.