Anisakiasis is one of the most common fishborne helminthic diseases in Japan, which is contracted by ingesting the larvae of the nematode Anisakis spp. carried by marine fish. Anisakis simplex sensu stricto (s.s.) and A. pegreffii are the dominant species in fish caught offshore Japan. The present study aimed to identify the anisakid species infecting Japanese patients and determine whether there is any difference in the pathogenetic potential of A. simplex (s.s.) and A. pegreffii. In total, 41 and 301 Anisakis larvae were isolated from Japanese patients and chub mackerel (Scomber japonicus), respectively; these were subjected to molecular identification using polymerase chain reaction targeted at a ribosomal DNA internal transcribed spacer region. Chub mackerel larvae were further examined for survival in artificial gastric juice (pH 1.8) for 7 days and for invasiveness on 0.75% solid agar over a 24-h interval. All clinical isolates, including those of asymptomatic, acute, and chronic infections as well as those from the stomach, small intestine, colon, and stool, were identified as A. simplex (s.s.). Chub mackerel harbored A. simplex (s.s.) and A. pegreffii larvae, together with a few larvae of other anisakid species. A. simplex (s.s.) larvae from chub mackerel tolerated the artificial gastric juice better than A. pegreffii, with 50% mortality in 2.6 and 1.4 days, respectively. In addition, A. simplex (s.s.) penetrated the agar at significantly higher rates than A. pegreffii. These results show that A. simplex (s.s.) larvae have the potential to survive acidic gastric juice to some extent and penetrate the stomach, small intestine, or colon in infected humans.