The influence of the groundwater level (GW), vegetable crop rotation and mineral fertilizers on the change in the composition of organic matter of medium-thick peat soil was determined. The study was conducted on lysimeters with an adjustable level (0.5; 1.0; 1.5 and 1–2.0 m) of groundwater occurrence; in a vegetable-feed crop rotation spread in time and space; under perennial grasses without prior cultivation of annual crops. It was established that in the arable layer (0.2 m) of peat soil, the bitumen content at GW level of 0.5 m was lower by 1.65% than at a depth of 1.0 m, and by 4.34% than at a depth of 1.5 m. The maximum amount of water-soluble substances (5.68%) and substances hydrolyzable by 2% HCl (34.25%) was established at GW level of 0.5 m. It was 1.2 and 3.4–3.8% higher than at groundwater level of 1–1.5 m, respectively. With GW level of 1.5 m, the amount of humic acids decreased by 4.5% compared to their occurrence at a depth of 0.5 m. The amount of fulvic acids in the arable layer depends on the GW level (r = 0.79). The content of substances hardly hydrolyzable by 80% H2SO4 in the arable layer decreased from 2.82 to 2.31% with an increase in groundwater depth from 0.5 to 1.5 m. It was observed that the amount of lignin decreased with an increase in groundwater depth from 0.5 (6.66%) to 1.5 m (5.30%). Five- year cultivation of crops in the vegetable-feed crop rotation did not lead to significant changes in the composition of the organic matter of peat. Grassing of peat soils with perennial grasses without sowing pre-crops ensures preservation of peat organic matter and prevents its substantial transformation. Mineral fertilizers reduce the rate of accumulation of hardly-hydrolyzable and non-hydrolyzable forms due to more severe oxidative-hydrolytic conditions in the soil and because of an increase in the amount of fresh plant material as a result of crop and root residues.