Polyunsaturated fatty acids such as n-3 and n-6 are known to influence reproductive performance of dairy cows. The objective of this study was to determine fatty acid (FA) profile in serum and follicular fluid of cows fed 3 different lipid diets as well as to determine the association between serum and follicular fluid in terms of FA concentrations. Nine nonlactating Holstein cows were randomly assigned to receive 1 of 3 diets supplemented with rolled oil seeds: canola (CAN = 3), sunflower (SUN = 3) or flax (FLX = 3) at 8%DM. After cows received their respective diets for at least 60 d, they were superovulated, inseminated and slaughtered 14 d after insemination. The experimental diets continued until 12 h before slaughter. Embryos and reproductive tissues collected were used in another study. Blood samples were collected on the day of insemination, and follicular fluid was collected from large ovarian follicles within 4 h of slaughter. Serum and follicular fluid were kept frozen until analyzed for FA concentrations. Fats from the follicular fluid and serum were extracted, methylated and FA profiles were determined by gas chromatography. Data were analyzed using the MIXED and CORR procedures of SAS. In follicular fluid, cows fed SUN had higher concentrations (mg/mL) of linoleic acid (0.60 vs. 0.30, 0.32) and total n-6 FAs (0.62 vs. 0.31, 0.33) and higher n-6/n-3 ratio (9.25 vs. 2.09, 3.84) than those fed either FLX or CAN. Similarly, cows fed FLX had higher concentrations of α-linolenic acid (C18:3; 0.14 vs. 0.08, 0.06), total n-3 FAs (0.16 vs. 0.09, 0.07) and higher n-3/n-6 ratio (0.49 vs. 0.10, 0.11) in follicular fluid than those fed either CAN or SUN. None of the FA other than γ-linolenic acid, in FLX cows, was altered by diet in serum. No significant correlations were observed between follicular fluid and serum FA profiles. Although diets supplemented with oilseeds selectively altered FA concentrations in follicular fluid, none of the major FA of relevance to reproductive function was altered in serum.