When laying hen diets are enriched with omega-3 polyunsaturated fatty acids to generate value-added eggs for human consumption markets, concentrations of alphalinolenic, eicosapentaenoic, and docosahexaenoic acids (ALA, EPA, and DHA) in the yolk can reach 250mg/50g whole egg. Flaxseed, a rich source of ALA, is commonly used for omega-3 enrichment; however, the impact of dietary flaxseed source (extracted oil vs. milled seed) on fatty acid transfer to egg yolk in laying hens is unknown. Therefore, transfer of ALA, EPA, and DHA into egg yolk from extracted flaxseed oil or milled flaxseed was evaluated in Hy-Line W-36 laying hens over an 8 wk feeding period (25 to 33 wk old). Hens (n=132) were randomly housed with 3 birds/cage (4 replicates/treatment) for each of the 11 treatment groups. Diets were isocaloric and consisted of a control diet, 5 flaxseed oil diets (0.5, 1.0, 2.0, 3.0, or 5.0% flaxseed oil), and 5 milled flaxseed diets (calculated flaxseed oil concentration from milled flaxseed 0.5, 1.0, 2.0, 3.0, 5.0%). Increasing dietary concentrations of flaxseed oil and milled flaxseed resulted in increased ALA, EPA, and DHA concentration in egg yolk, and fatty acid deposition from flaxseed oil was 2 times greater compared to milled flaxseed when fed at the same dietary inclusions (P