This work investigated the effects of feeding whole pomegranate by-product (WPB) to lambs on ruminal, liver and intramuscular fatty acids (FA). Seventeen lambs, divided into two groups, were fed for 36 days with a cereal-based concentrate diet (CON) or with a concentrate diet containing 200 g/kg DM of WPB to partially replace barley and corn (WPB). The dietary treatment did not affect the final body and carcass weight, the dry matter intake or the average daily gain. However, total polyunsaturated FA (PUFA), linolenic, rumenic (RA) and vaccenic (VA) acid were increased in liver (+15%, +32%, +344% and +118%, respectively) and muscle (+46%, +38%, +169% and +89%, respectively) of WPB lambs (P < 0.05). Punicic acid and three isomers of conjugated linolenic acid were detected exclusively in the rumen and tissues of WPB-lambs. The C18:1 t10/t11 ratio in rumen digesta or in tissues was reduced by feeding WPB (-791%, -690% and -456%, respectively in rumen, liver and muscle; P < 0.001), suggesting that the WPB prevented the t10-shift rumen biohydrogenation pathway. In conclusion, the inclusion of WPB into a concentrate-based diet can be a strategy to improve the FA composition of meat, without effects on the animal performances.