There has been a marked decline in the fertility of dairy cows over the past decades, and metabolomic analysis offers a potential to investigate the underlying causes. Metabolite composition of the follicular fluid, which presents the intrafollicular environment, may be an important factor affecting oocyte maturation and subsequent early embryo development. The aim of the present study was to investigate the metabolic differences between follicular fluid from the dominant follicle of lactating cows and heifers using gas chromatography mass spectrometry (GC-MS)-based metabolomics. Follicular fluid and serum were collected from cows and heifers over three phases of follicle development: newly selected dominant follicles, preovulatory follicles prior to oestrus and post-LH surge follicles. Analysis of the fatty acids revealed that there were 24 fatty acids and 9 aqueous metabolites significantly different between cows and heifers. Of particular interest were the higher concentrations of saturated fatty acids (palmitic acid, P=0.001; stearic acid, P=0.005) in follicular fluid from cows and higher docosahexaenoic acid levels (P=0.022) in follicular fluid from heifers. Analysis of the metabolite composition of serum revealed that follicular fluid had a unique lipid composition. The higher concentrations of detrimental saturated fatty in cows will have a negative impact on oocyte maturation and early embryo development. Overall, the results suggest that the follicle microenvironment in cows potentially places their oocytes at a developmental disadvantage compared with heifers, and that this may contribute to well-characterised differences in fertility.