Seasonal change of milk composition could offer opportunities for dairy manufacturers. A systematic survey on seasonal variation of six classes of polar lipids was conducted with 19 Holstein cows over the entire milking season using liquid chromatography-mass spectrometry technique. This study revealed that most polar lipid classes were positively correlated with the total fat content, but negatively correlated with fat globule size. All polar lipid classes displayed a large cow-to-cow variation as well as seasonal variation. All of the six classes showed a gradual increase over the milking season with the highest concentration observed in May (autumn). However, the proportion of different polar lipid classes remained constant during the entire milking season. This finding suggests that the production of polar lipids is highly regulated in the mammary gland. The implication of such a seasonal variation of polar lipids in the nutritional and technological aspects of milk is discussed.