In agriculture, although it is important to identify species of pest insects, the morphological identification is often difficult. DNA genotyping is useful for the identification of species in morphologically indiscriminable species. Thrips tabaci (Lindeman) can be divided into two reproductive forms (arrhenotoky and thelytoky, each of which different in pesticide resistance) but morphological discrimination is not possible. Here, we establish a simple method to discriminate the strains based on their mitochondrial DNA sequences. Phylogenetic analysis including the T. tabaci and congeneric species provided ancestor sequences of each strain of T. tabaci. Based on the ancestor sequences, we developed a primer set that include strain specific primers on sense strand and common primer on anti sense strand. Using this primer set, the strains of 196 individuals of T. tabaci were successfully assigned to each of genotypic forms. As the phylogeny and ancestor sequences were based on worldwide samples, this method will work well on most populations around the world.