The aim of this study was to compare the fatty acid profile of intramuscular fat (IMF) in female roe deer (Capreolus capreolus L.) and red deer (Cervus elaphus L.) living in the wild. The experimental materials comprised samples of the Longissimus lumborum (LL) muscle collected from 20 carcasses of does aged 3–5 years and 15 carcasses of hinds aged 4–6 years. All animals were hunter-harvested in the forests of North-Eastern Poland. The IMF of does had considerably higher concentrations of monounsaturated and polyunsaturated fatty acids. Therefore, it was characterized by more desirable values of quality parameters, and provided more health benefits. The observed differences in the fatty acid composition of IMF between does and hinds are important in view of both the nutritional value of meat and its susceptibility to lipid oxidation and rancidification.