Fatty acid composition with a special stress on polyunsaturated fatty acids (PUFA) was studied in the sn-2 location of tuna phospholipids (PL) in eight commercial species. Comparison with the sn-1 location and changes of both positions (sn-1 and sn-2) during cooking were also analyzed. When the sn-1 and sn-2 locations in albacore tuna were compared, some differences were noted. The sn-1 position showed higher contents in saturated (16:0 and 18:0) and 18:1 omega 7 fatty acids; in contrast, higher proportions in PUFA (22:6 omega 3, 20:5 omega 3, and 20:4 omega 6) and in 18:1 omega 9 were obtained in the sn-2 location. The comparative study of the sn-2 position of PL showed a similar pattern of distribution in the different tuna species. In all cases the main fatty acid was 22:6 omega 3, followed by 18:1 omega 9, 20:5 omega 3, 16:0, 20:4 omega 6, and 24:1 omega 9. A high omega 3/omega 6 ratio was found in all cases, although great differences were noted between the highest (bonito and yellowfin) and the lowest (little tunny and skipjack) values; ratios between the most abundant PUFA belonging to the omega 3 and omega 6 series (22:6 omega 3/20:4 omega 6 and 20:4 omega 6/20:5 omega 3) showed similar differences. The behavior of both positions (sn-1 and sn-2) in albacore PL was studied during cooking. As a result of the thermal treatment, some changes in the fatty acid composition were seen in both locations. The sn-2 position showed a decrease in 22:6 omega 3 and an increase in 18:1 omega 9. On the contrary, a decrease in 18:0 was obtained in the sn-l position, along with an increase in 22:6 omega 3 and 20:4 omega 6.