Perineal glands in guinea pigs are part of the sebaceous glandular complex. Their secretions are used for scent marking. This is important for social status and can be seen in both sexes and castrated males. Discrepancy exits about the existence of these glands in female guinea pigs and knowledge of the anatomical consequences of castration on the male perineal glands is sparse. To examine these uncertainties related to gender, perineal glands from 13 sexually mature pet guinea pigs were examined macro- and microscopically. Clear gender differences in the anatomy of perineal glands were found, and castrated males showed signs of atrophy and fatty infiltration in the glands. Females do have perineal glands, although smaller than the glands in the male. The glands are typically sebaceous with multiple excretory ducts. A macroscopic unique feature in the males was the clearly evident orifices of a large excretory duct on each side of the slightly everted perineal sac. However, the reason for this gender difference is not clear. In castrated males, the orifices were atrophied and difficult to see. In addition, the sebaceous glands of the hair follicles in the skin folds of the perineal opening were smaller and less abundant in females and castrated males. The changes in castrated males are presumably linked to the hormonal changes and decreased secretion after castration. The dense keratin layer in the perineal sac was thicker in males than in both castrated males and females and could contribute to the concrement formation seen mainly in males.