Anisakis simplex complex presently comprises three sibling species, A. simplex sensu stricto, A. pegreffii and A. simplex C. A. simplex is a common parasite in fishes and cephalopods and capable of causing anisakiasis in humans. Therefore, identification of sibling species of A. simplex was important for human health. In this study, one hundred Anisakis type I larvae isolated from eighty five patients with anisakiasis in Hokkaido and Kyushu in Japan were analyzed by adapting the new molecular method that can identify the sibling species of A. simplex complex. Based on the restriction fragment length polymorphism (RFLP) pattern of ITS regions including 5.8 subunit rRNA gene, we identified two sibling species, A. simplex s. str. and A. pegreffii. However, the infection rate of A. simplex s. str. was significantly higher than that of A. pegreffii. Eighty four (98.8%) out of the eighty five patients were infected with A. simplex s. str. On the contrary, one patients (1.2%) in Kyushu infected with A. pegreffii. This study provided basic information about human infection with A. simplex complex. Furthermore, we suggested that A. simplex s. str. is the most important etiological agent in Japan.