Here we present a comparison of the different chromosome preparation methods, including the method of the single embryo or larva, the method of juvenile-swimming and the method of phytohemagglutinin injection, used for the different developmental stages of the half-smooth tongue sole, Cynoglossus semilaevis. The mean index of mitosis for the three methods was 0.79%, 0.09% and 0.15%, respectively. From the well-spread metaphase chromosomes obtained, it was determined that C. semilaevis has a diploid chromosome number of 42 and heterotypic sex chromosomes. Also, the metaphase chromosomes produced could be used for karyotype preparation and banding studies. The three methods are also successfully used for chromosome preparation in Japanese flounder (Paralichthys olivaceus) and summer flounder (Paralichthys dentatus).