Alfalfa (Medicago spp.) plays an important role in a farming system and contributes to stabilization of the ecosystem by improving soil physical, chemical and biological properties. However, the use of alfalfa is limited due to its susceptibility to edaphic conditions. The objective of this study was to evaluate the agro-biological traits of alfalfa under different soil conditions. Field experiments were carried out in two locations with optimised management. Agro-biological traits of 46 alfalfa (Medicago sativa L. and M. varia Marth.) accessions of different geographic origin were assessed in a high productivity Endocalcari Epigleyic Cambisol. Although environmental conditions were favourable for alfalfa development and growth, the tested genotypes significantly differed in agro-biological traits: plant height of regrowth in spring and after cuts and at flowering period as well as seed yield. Cultivars from colder climate countries (Estonia, Latvia and Lithuania) were the least affected by the downy mildew. In a low productivity acidic Bathygleyic Dystric Glossic Retisol, the pH had a major influence on the distribution of the root system of alfalfa-grass mixture. During the first experimental year, in the naturally highly acidic soil (pH 3.9), where the mineral nutrition was unfavourable, alfalfa produced longer roots, their diameter and mass were larger than in the soil with a pH of 5.0. In the third year of cultivation, in the naturally highly acidic soil only 9.1% of alfalfa plants had survived. A strong correlation (r = 0.873, P < 0.01) was determined between the amount of alfalfa in the dry matter yield and soil acidity.