The objective of the study was to determine the content of fatty acids (FAs) in subcutaneous fat from tailhead area of heavy lambs of Berrichon du Cher (BE) and Suffolk (SF) breeds. Lambs of both sexes were kept in semi-intensive production systems (SI1 and SI2). The production systems differed in nutrition during period of two weeks before slaughter: BE/SI1 lambs were fed with hay and concentrates, SF/SI2 lambs were on pasture and dam milk. The analysis of variance with two factors − breed/production system (BE/SI1, SF/SI2) and lamb sex (males, females) was applied to investigate differences between lamb groups. The content of essential FAs (sum of linoleic and α-linolenic acids), determined by a gas chromatography method, was higher (P < 0.001) in subcutaneous fat of BE/SI1 lambs (2.90 g.100 g-1 FAME) when compared to SF/SI2 lambs (2.53 g.100 g-1 FAME). The contents of arachidonic and docosapentaeonic acids were higher (P < 0.05) in BE/SI1 lambs (0.08 and 0.15 g.100 g-1 FAME) than in SF/SI2 lambs (0.05 and 0.12 g.100 g-1 FAME), whereas the contents of eicosapentaeonic and docosahexaenoic acids did not differ between groups. The content of conjugated linoleic acid (CLA) was higher (P < 0.001) in SF/SI2 lambs than in BE/SI1 lambs (1.84 and 1.09 g.100 g-1 FAME). In relation to sex, the content of CLA in ewe lambs (1.60 g.100 g-1 FAME) was higher (P < 0.05) than in ram lambs (1.33 g.100 g-1 FAME). The content of docosapentaeonic acid was also higher (P < 0.05) in ewe lambs (0.16 vs. 0.12 g.100 g-1 FAME). These findings contribute to expanding knowledge about sheep breeds in Slovakia.