Miniature swine (minipigs) are often used in non-rodent toxicity studies. However, unlike other animal species, the parathyroid glands of minipigs are often covered with thymic tissue and are similar in color, making macroscopical identification difficult. We investigated a method for sampling and tissue preparation of the parathyroid glands using 5- to 7-month-old minipigs. The glands were identified by finding the insertion site of a branch from the carotid artery into the cervical part of the thymus. Then the glands were marked and sampled. In a preliminary study, the glands were macroscopically and microscopically detected in 3/8 animals. The glands were not identified macroscopically in 5/8 animals but were detected in 3 of these animals. In total, we succeeded in detection of the glands in 6/8 animals (75%). The method was applied in the main toxicity study, and we succeeded in 100% detection through technical advancement. The method described herein enables high rate of detection and is useful in the pathological evaluation of the parathyroid glands of minipigs.