As a typical seasonal breeding animal, male muskrats have a pair of scent glands that can emit musky odor substances to attract females during the breeding period. The present study aimed to visualize the differences in the distribution of lipids in the scent glands of muskrats during their different reproductive statuses by imaging mass spectrometry and quantitative real-time PCR (qRT-PCR). The results revealed remarkable differences in the expression and spatial distribution of lipids detected in the scent glands of muskrats during the different reproductive statuses. In addition, the expression levels of lipid molecules PC (32:0) and LysoPC (16:0) were found to be significantly higher in the breeding season than in the non-breeding season. Moreover, the mRNA expression levels of lipid synthesis enzyme Pemt and Pla2g4b were higher in the breeding season than in the non-breeding season, and there were positive correlations between the expression intensities of lipid molecules and the expression levels of Pemt and Pla2g4b. The present study investigates the changes and distribution of the endogenous lipid in the scent glands of muskrats and elucidates that the seasonal changes in the lipid metabolism may affect the functions of the scent glands in muskrats.