Identification of heterotic groups and patterns among breeding populations provides fundamental information to help plant breeders more knowledgeably manipulate heterosis. A diallel analysis was conducted among nine alfalfa ( Medicago sativa L.) germplasms, commonly referred to as African, Chilean, Flemish, Indian, Ladak, M. falcata, M. varia, Peruvian, and Turkistan, which represent a significant proportion of the genetic diversity present in US cultivars. Heterotic responses were determined by evaluating forage yield of the germplasms and their 36 half-diallel hybrids in seeded plots that were harvested five times in each of 2 years. Commercially acceptable yields were obtained from some hybrids of unimproved parents, where at least one parent was adapted to the study environment. Variation among crosses was attributed primarily to general combining ability (GCA) effects; however, specific combining ability effects were also significant. GCA estimates for African, Chilean and Peruvian were positive, while those for Ladak, M. falcata, and M. varia were negative. Estimates for variety heterosis effects were positive for Peruvian and M. falcata and negative for Indian and M. varia. Significant mid-parent heterosis [(MPH) range of -21% to 55%] and high-parent heterosis [(HPH) range of -33% to 23%] was detected. M. falcata hybrids exhibited the highest MPH values. However, this likely reflects the poor yield of M. falcata per se in the study environment and consequently, low MPH values. Peruvian hybrids demonstrated the highest cross mean performance, significant positive MPH in all crosses, and positive HPH in five out of eight crosses. The results indicate that Peruvian should be recognized as a heterotic group. Alfalfa breeders may wish to explore opportunities for heterotic yield gains that are likely to exist in hybrids between the Peruvian germplasm and elite breeding populations, in particular, those adapted to the southwestern United States. MPH results suggest that alfalfa breeders may have capitalized on the heterotic response between Flemish and M. varia during past development of alfalfa synthetics adapted to the central and northern latitudes of the United States.