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To evaluate the agro-biological traits and select the promising populations of alfalfa under pedoclimatic conditions, field experiments were carried out in two locations of the Lithuanian Research Centre for Agriculture and Forestry (western Lithuania 55°70′ N, 21°49′ E and central Lithuania 55°23′ N, 23°51′ E). Eleven populations were established in acidic soil with a pH of 4.0 (experimental site I) and neutral soil with a pH of 7.0 (experimental site II). The productivity and agro-biological traits: fresh matter yield, dry matter yield, seed yield, stem thickness, and stem, leaf, and inflorescence weight of alfalfa populations were evaluated from 2019 to 2021. In the acidic soil, the populations 3056, 3132, 3130, and 3058 yielded more fresh and dry matter compared to the standard cultivar Birutė in the period from 2019 to 2021. In the acidic soil, populations 3131 and 3057 had a higher seed yield compared to the cultivar Birutė in 2019 and 2020, respectively. The 3086 population significantly differed in leaf weight compared to the cultivar Birutė. The cultivar Birutė and population 3132 were similar in stem thickness and fresh matter yield. In the neutral soil, population 3056 yielded more fresh matter compared to the cultivar Birutė. [ABSTRACT FROM AUTHOR]