Alfalfa is planted in more than 30 million hectares worldwide, but despite its popularity in temperate regions, it is not widely grown in subtropical agroecosystems. It is critical to improve alfalfa for such regions, considering current predictions of global warming and the increasing demands for animal-based products. In this study, we examined the diversity present in subtropical alfalfa germplasm and reported genetic parameters for forage production. An initial screening was performed from 2014 to 2016, evaluating 121 populations from different subtropical origins. Then, a breeding population was created by crossing selected plants, resulting in 145 full-sib and 36 half-sib families, which were planted in a row-column design with augmented representation of three controls (‘Bulldog805′, ‘FL99′ and ‘UF2015′). Dry matter yield (DMY), canopy height (AH), and percentage blooming (BLOOM) were measured across several harvests. Moderate narrow-sense heritability and high genetic correlations between consecutive harvests were estimated for all traits. The breeding line UF2015 produced higher DMY than FL99 and Bulldog805, and it could be a candidate cultivar release. Several families produced higher DMY than all checks, and they can be utilized to develop high yielding and adapted alfalfa cultivars for subtropical agroecosystems.