Hippocampus abdominalis are an economically important aquatic species in China. To understand the genetic structure of five populations of the main farmed distended seahorses in China [the third cultured stock in Shangdong province (SD), the first cultured stock in Fujian province (FJ1), the sixth cultured stock in Fujian province (FJ6), the 12th cultured stock in Tai Wan (TW), and breeding offspring of one Australian wild species (AS)], We developed six polymorphic microsatellite markers of the H. abdominalis, and used these 6 microsatellite markers for genetic analysis of five populations of H. abdominalis. The results showed that 63 alleles were detected by 6 pairs of primers in 144 samples. The number of alleles ranged from 8 to 14, with an average of 10.5 and PIC＞0.25, indicating that all 6 microsatellite loci had high polymorphism. In addition, the observed heterozygosity and expected heterozygosity of the five populations were 0.6603 and 0.8104, respectively, indicating that the genetic diversity of the five populations was relatively rich. Further, the molecular variance analysis and genetic analysis was found that the genetic variation of the five populations mainly came from within the population, and a relatively high level of genetic distance was maintained among the 5 populations, among which the genetic distance between Taiwan (TW) and Fujian (FJ1) populations was the largest at 0.4529. In summary, there were genetic differences among the five H. abdominalis populations, and the genetic diversity of the populations was relatively rich and there was no decline in genetic diversity, indicating the 5 H. abdominalis populations could continue to be used for subsequent breeding.