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Alfalfa (Medicago sativa L.) is a valuable crop worldwide. In addition to use as forage, the nitrogen fixation capabilities of root nodules make it a valuable component in maintaining soil health. Unfortunately, the semi-arid region of the Northern Great Plains introduces a number of environmental stresses that are detrimental to stand establishment and persistence. This study aimed to identify alfalfa populations possessing the ability to produce adventitious shoots from root segments. The ability to produce adventitious shoots from roots is generally considered a favorable trait for increasing stress resistance. Seven Plant Introductions (PIs), selected from the National Plant Germplasm System, and one commercial cultivar (Persist II) as a control, were evaluated. Two six- centimeter root segments, originating at one and seven centimeters below the cotyledonary node, were cut from one year old plants. Root segments were planted in Miracle-Gro (R) potting soil, and cultivated under greenhouse conditions for 16 weeks. Adventitious shoot emergence was recorded throughout the experimental period. At the end of the experimental period, the number of adventitious shoots, shoot survival, flowering status, and non-shoot-bearing root segment decay were determined. Six of the seven PIs produced adventitious shoots. No shoots were produced by Persist II and PI494662. Among the six populations, the frequency of generating adventitious shoots ranged from 3.8% to 57%. Shoot survival ranged from 33% to 100%. Speed of regeneration from roots and viability of root segments in soil varied among populations. PI631678 had the greatest frequency and speed of regeneration from roots, as well as high survival and flowering rates. Adventitious shoot regeneration and survival was not affected by origin distance below the cotyledonary node within in the same populations.