Amplified fragment length polymorphism (AFLP) analysis was used to evaluate the genetic diversity of four wild geographical populations of Japanese flounder (Paralichthys olivaceus). A total of 775 loci (58.32% of which was polymorphic) in the range between 100 and 1 300 base pairs were detected from 110 individuals using seven primer combinations. The percentage of polymorphic loci detected by single primer combination for each population was calculated, ranging from 19.59% to 53.33%. Genetic similarities within and among the populations were calculated from the binary matrices of presence-absence. Phylogenetic tree of four populations was constructed by using the UPGMA method using PHYLIP Version 3.5. According to intrapopulation genetic similarities, CW population displayed the highest genetic diversity value and KY population had the lowest genetic diversity value. The distance between CW and CF populations was the farthest, which was possibly resulted from the farthest distance of Weihai of Shandong and Fujian of China compared with the geographical distance between other locations of populations. The subpopulation differentiation value (G_ st ) is 0.356 5, showing a certain extent of differentiation among the four geographical populations. AFLP technology was confirmed to be an effective tool to assess within-and among-population genetic diversity of Japanese flounder. The present survey provided significant insights for research in the Japanese flounder breeding program. Amplified fragment length polymorphism (AFLP) analysis was used to evaluate the genetic diversity of four wild geographical populations of Japanese flounder (Paralichthys olivaceus). A total of 775 loci (58.32% of which was polymorphic) in the range between 100 and 1 300 base The percentage of polymorphic loci detected by single primer combination for each population was calculated, ranging from 19.59% to 53.33%. Similarity within and among the populations were calculated from the binary matrices of presence -absence. Phylogenetic tree of four populations was constructed by using the UPGMA method using PHYLIP Version 3.5. According to intrapopulation genetic similarities, CW population displayed the highest genetic diversity value and KY population had the lowest genetic diversity value. The distance between CW and CF populations was the farthest, which was possibly resulted from the farthest distance of Weihai of Shandong and Fujian of China compared with the geographical distance between other locations of populations. The subpopulation differentiation value (G_st) is 0.356 5, showing a certain extent of differentiation among the four geographical populations. AFLP technology was confirmed to be effective tool to assess within-and among-population genetic diversity of Japanese flounder. The present survey provided significant insights for research in the Japanese flounder breeding program.