This study investigated the effects of genotype and diet on meat fat composition and palatability obtained from Alentejana (AL) and Barrosã (BA) breeds. Herein, 20 males from each breed allocated at 11 months of age were fed ad libitum a low-forage diet or a high-forage diet and slaughtered at 18 months of age. Trained sensory panel analysis found that the longissimus lumborum (Ll) muscle from BA had higher tenderness, juiciness and overall acceptability scores than the AL breed. The highest scores for those attributes were observed in the BA breed fed the high-forage diet. Regarding the semitendinosus (St) muscle, breed was a source of variation of tenderness scores. In contrast to the Ll muscle, the highest tenderness scores for the St muscle were observed in the AL breed. The intramuscular fat (IMF) content was positively correlated with tenderness, juiciness and overall acceptability in Ll muscle and negatively correlated with flavour in the St muscle. The levels of 14:0 and 16:0, 16:1c9, 18:1c9 and 18:1c11 were positively correlated to juiciness, tenderness and overall acceptability in the Ll muscle. These correlations were not observed in the St muscle, which may be related to its low IMF content. Nonetheless, negative correlations were observed for the St muscle between flavour and 14:0, 16:0 and 18:0 FA contents.The IMF varied widely in the Ll but not in the St muscle. The latter had higher levels of 16:1c9 and trans fatty acids (∑TFA) in the BA than in the AL breed. Regarding the Ll muscle, the BA had higher amounts of 14:0, 16:0, 16:1c9, 18:0, 18:1c9, 18:1c11, saturated fatty acids (∑SFA), cis monounsaturated fatty acids (∑cis MUFA), ∑TFA and n-3 polyunsaturated fatty acids (∑n-3 PUFA) than the AL breed. The diet exerted an influence on the IMF content and on the levels of 14:0, 16:0, 16:1c9, 18:0, 18:1c9, 18:1c11, ∑SFA, ∑cis MUFA and ∑TFA in both Ll and St muscles. Moreover, the levels of ∑n-3 PUFA in the Ll muscle and 18:2n-6, 20:4n-6, ∑n-6 PUFA and ∑PUFA in the St muscle were influenced by diet. The results obtained in this study, with two Portuguese breeds, confirm that genetic background plays a major role in the determination of meat eating quality.