Abstract: To understand the current population genetic structure of half-smooth tongue sole and explore the trend and status of effective population size (N_e) in half-smooth tongue sole, in this paper, 800 4-month-old half-smooth tongue sole were identified by gender-specific genetic markers, and 297 genetic females were selected. Then64 416 SNP markers were obtained by simplified genome sequencing method (2b-RAD). The genome-wide linkage disequilibrium (LD) was used to obtain the linkage disequilibrium distribution on each chromosome. The effective population size was calculated based on the different physical distances between the markers for each historical generation. In order to analyze the genetic structure of the population of half-smooth tongue sole selected by natural and artificial means, the sizes of 7 different chromosomal fragment lengths were detected. And the results showed that the effective population size two generations ago was only about 29. The high-throughput 2b-RAD sequencing method was first applied to the estimation of the effective population size of aquatic animals, which provided a reference for the breeding work of other aquatic species. The effective population sizes decreased when the historical generation was closer. It is important for half-smooth tongue sole breeding and conservation to estimate the effective population size for current generation.