The pathogenic potentials of two sibling nematodes Anisakis simplex sensu stricto (s.s.) and A. pegreffii were compared by in vitro and in vivo studies. Live third-stage larvae of each species were subjected to agar blocks made using PBS or RPMI-1640, overlaid with different supernatants (artificial gastric juice, PBS, and RPMI-1640), and their penetration ability was compared. Their tolerance of artificial gastric juice was also tested. Further, they were introduced into rats by gastric intubation, and the in vivo locations of them were investigated. A. pegreffii showed higher penetration ability than A. simplex (s.s.) in most of the experimental conditions, except for the RPMI-1640 agar block overlaid with artificial gastric juice. In an acid tolerance test, the mean survival times were 6.1 days for A. simplex (s.s.) and 4.2 days for A. pegreffii. In an animal experiment, A. simplex (s.s.) stayed for a shorter time in the stomachs of rats than A. pegreffii. Some A. pegreffii and A. simplex (s.s.) were embedded in the gastric mucosa or freely existed in the abdominal cavity. All of these results suggest that A. pegreffii has the pathogenic potential to cause anisakidosis in humans when ingested, as does A. simplex (s.s.).