Alfalfa is the most important forage legume in the production of voluminous fodder. Although not primarily produced for its seeds, the seed yield is still important for the recognition and commercial viability of the cultivars on the market. Creating a cultivar of superior yield and forage quality with satisfactory seed production is one of the biggest challenges for alfalfa breeders and seed producers. The objective of this study was to determine forage and seed yields of 19 newly developed alfalfa experimental populations (ABP 1–19) of the Agricultural Institute Osijek during a long-term research period (2014–2018) in different climatic conditions. Significant differences were found between ABPs and years for forage and seed yields. Three-year (2014–2016) average green mass yield ranged from 68.41 t ha–1 (ABP 6) to 78.05 t ha–1 (ABP 19) and dry matter yield from 13.73 t ha–1 (ABP 6) to 15.30 t ha–1 (ABP 18). The average two-year (2017–2018) seed yield varied from 150.78 kg ha−1 (ABP 9) to 335.35 kg ha−1 (ABP 7). Annual forage yield significantly increased from the year of establishment to the second and third growing seasons of alfalfa. The highest average annual yield of green mass (90.24 t ha−1) was achieved in 2015, dry matter yield (17.62 t ha−1) in 2016 and the seed yield (394.17 kg ha−1) in 2017. During the researched period there was a considerable decreasing trend in forage yield from the first to the last cut, except in the year of the alfalfa establishment. Several alfalfa populations (ABP 19, 8, 14) superior in all analyzed traits were identified, and they represent top performing materials with the potential for developing and releasing cultivars in the near future. Populations with high yields of green mass and dry matter (ABP 12, 18, 1) and seed yield (ABP 7, 4) were also detected and represent valuable genetic material to improve our alfalfa breeding program.