The aim of the work was to assess the influence of the locality on the nutrient content of alfalfa, grown in five consecutive years 2018–2022. The Troubsko location belongs to the beet production area, with an altitude of 270 m, the Hrušovany nad Jevišovkou location is located in the Dyjsko-Svrateck valley near the river Dyje, and has an average altitude of 181 m. In each of the two locations, alfalfa samples were taken from the first three cuttings, which were subsequently chemically analyzed by conventional methods according to AOAC (2005). Troubsko is an area with a lower average temperature and a lower amount of precipitation, which is also why alfalfa was harvested there about 2 days later. Compared to the Hrušovany nad Jevišovkou location in Troubsko, alfalfa was determined to have a higher content of dry matter, ADF, NDF and ash and a lower content of N-substances at the time of harvest.