Abstract Cereals are an important source of vitamins, minerals, dietary fibre, and various phytochemicals, such as plant lignans. Lignans are a group of phenolic compounds that are commonly found in plants as their secondary metabolites. Lignans, found also in foodstuffs such as grains and seeds, are biologically active antioxidants, which reduce the occurrence of diabetes and cardiovascular diseases and prevent the progression of breast and prostate cancer. The aim of the study was to determine concentrations of lignans in different barley and oat genotypes grown under organic and conventional farming practices. Samples were collected in 2013 und 2014. The concentration of lignans was determined by high-performance liquid chromatography coupled with tandem mass spectrometry (HPLC-MS/MS). The highest concentration of total lignans was found in oat grains: 507.1 ± 3.0 (509.2 in 2013 and 505.0 in 2014) µg·100−1g. In barley grains, total lignin concentration was 425.2 ± 30.3 (403.8 in 2013 and 446.6 in 2014) µg·100−1g were detected. Concentration of some lignans, like HO-Mata, Lari and Pino, was higher both in oat (450.0, 3323 and 1159.8 µg·kg−1, respectively) and barley grain (334.3, 3297 and 334.4 µg·kg−1, respectively). The lignan concentration in studied oat and barley samples did not differ significant depending on cultivation practice.