The objectives of this study were to investigate the polar and neutral lipid fatty acid composition, content of retinol, α-tocopherol, γ-tocopherol and level of oxidation of the pig muscle (M. longissimus dorsi). Female and castrated male Hampshire crossbreeds were produced in two systems. One group was raised indoors with a more polyunsaturated diet and the other raised outdoors with a more saturated diet. The level of polyunsaturated fatty acids in muscle was higher in the indoor females compared with the outdoor females, indoor castrated males and outdoor castrated males. The increased level of polyunsaturated fatty acids in the muscle, which was accompanied by a relatively low content of α-tocopherol, increased the susceptibility to lipid oxidation in the form of MDA (malondialdehyde) in the indoor female pigs. Finally, the level of polyunsaturated fatty acids in the polar lipids was affected by the RN genotype, and this difference was dependent on sex. In conclusion, diet has a major effect on the fatty acid composition and oxidation stability in pork muscle, but additional factors such as sex and RN genotype might also contribute.