The effect of a 10-week supplementation with polyunsaturated fatty acids [via sunflower oil/DHA-rich algae (SUNA) or linseed oil/DHA-rich algae (LINA) enriched diets] versus saturated fatty acids (SAT) of lactating German Holstein dairy cows in mid-lactation on expression patterns of lipid metabolism-associated genes and gene products in hepatic, longissimus muscle and subcutaneous/perirenal/omental adipose tissue was assessed. Most pronounced transcriptomic responses to dietary PUFA were obtained in hepatic [down-regulated ACACA (FC = 0.83, SUNA; FC = 0.86, LINA), FADS1 (FC = 0.60, SUNA; FC = 0.72, LINA), FADS2 (FC = 0.64, SUNA; FC = 0.79, LINA), FASN (FC = 0.64, SUNA; FC = 0.72, LINA), SCD (FC = 0.37, SUNA; FC = 0.47, LINA) and SREBF1 (FC = 0.79, SUNA, LINA) expression] and omental adipose [up-regulated ACACA (FC = 1.58, SUNA; FC = 1.22, LINA), ADFP (FC = 1.33, SUNA; FC = 1.32, LINA), CEBPA (FC = 1.75, SUNA; FC = 1.40, LINA), FASN (FC = 1.57, SUNA; FC = 1.21, LINA), LPL (FC = 1.50, SUNA; FC = 1.20, LINA), PPARG (FC = 1.36, SUNA; FC = 1.12, LINA), SCD (FC = 1.41, SUNA; FC = 1.17, LINA) and SREBF1 (FC = 1.56, SUNA; FC = 1.18, LINA) expression] tissue. Interestingly, gene/gene product associations were comparatively low in hepatic and omental adipose tissue compared with longissimus muscle, perirenal adipose and subcutaneous adipose tissue, indicating matches only in regard to minor concentrations of SCD product 18:1c9, FADS1 product 20:4n-6 and FADS2 product 18:3n-6 in hepatic tissue, and higher concentrations of ACACA and FASN gene products 12:0 and 14:0 and SCD product 18:2c9,t11 in omental adipose tissue. Whereas all analyzed tissues accumulated dietary PUFA and their ruminally generated biohydrogenation products, tissue-divergent preferences for certain fatty acids were identified. This descriptive study reports tissue-divergent effects of dietary PUFA and outlines the significance of a PUFA intervention with regard to dairy cows' nutritional management.