Knowledge of the genetic structure of ecologically important species provides insight into population dynamics and persistence, which is important for decisions concerning ecosystem conservation and management. Seagrass ecosystems are being degraded in China due to coastal anthropogenic disturbances like eutrophication and pollution, but their genetic ecology is still poorly understood. In this study, we collected Enhalus acoroides samples from three lagoons and five offshore open-water sites along the east coast of Hainan Island to investigate its genetic diversity and structure using ten microsatellite loci. A total of 66 alleles were found, and the genetic diversity indices (i.e. mean number of alleles per locus, allelic richness and heterozygosity) varied among the eight populations. Assignment tests showed that the E. acoroides populations consists of three genetic clusters. Impeded gene flow among lagoonal populations was found, while connectivity existed among the open-water populations. This pattern seemed to be shaped by geographic isolation and ocean currents. Based on the genetic contribution analysis, we recommended that the E. acoroides populations at Tielugang and Gangdong need protection priority given they contribute higher genetic diversity, but are currently high risk populations under threats of pollution and physical disturbance.