Polyunsaturated fatty acid (PUFA) deposition in lambs’ muscles could be influenced by their mothers’ diet. The aim was to study the profile of fatty acids in the muscle of lambs from ewes supplemented with different sources of PUFA to achieve a healthier meat for the consumer. On day 100 of gestation, pregnant ewes grazed on natural grass were divided into three groups (n = 20) and supplemented with PUFA: Group A: 700 g of a ration rich in PUFA, Group B: 700 g of a ration for sheep + 20 mL of fish oil and Group C: 700 g of the same ration. After parturition, each group was subdivided: ten ewes continued with the same diet until the end of lactation; the other ten were fed only natural grass. The values of n-3 and n-6 PUFA and alpha-linolenic acid (ALA), eicosapentaenoic acid (EPA), docosahexaenoic acid (DHA), linoleic acid (LA) and arachidonic acid (AA) were analyzed in Longissimus lumborum muscles of lambs at 90 and 120 days of life. The feeding of ewes during lactation favorably influenced the lipid profile of the lamb muscle, increasing the concentration of n-3 and n-6 PUFA, ALA, LA, AA, DHA and EPA. The supplementation of ewes with fish oil and/or a ration rich in PUFA improved the LA/ALA ratio in lambs’ meat.