Human milk oligosaccharides (OS) play an important role in protecting the neonate. In addition to fructo-oligosaccharides and galacto-oligosaccharides, bovine milk OS have great potential to be used in pediatric food products to mimic the functions of human milk OS. Currently, little is known about the accumulation of OS in bovine milk in relation to genetic and environmental factors. A systematic survey on seasonal variation of 14 major OS was thus conducted with 19 cows over the entire milking season using a liquid chromatography-mass spectrometry technique. This study revealed a number of significant correlations between structurally related and structurally nonrelated OS and a substantial individual animal difference for all 14 OS. Most of the 14 OS displayed a remarkable seasonal variation in abundance (up to 10-fold change), with the highest abundance observed in April and May (i.e., autumn) for the majority of the 19 cows.