A method of in vitro plant regeneration for both the selenium-hyperaccumulator Astragalus racemosus ‘Cream Milkvetch’ and the nonaccumulator Astragalus canadensis ‘Canadian Milkvetch’ was developed with two induction media, M1 and M2. The M1 and M2 contain Murashige and Skoog basal medium plus vitamins, 8.07 μm N-(2-chloro-4-pyridyl)-N′-phenylurea, 2.5% (w·v−1) sucrose, 0.7% (w·v−1) agar (pH 5.7), and 0.89 μm or 3.12 μm a-naphthaleneacetic acid, respectively. In vitro cultures were initiated on these two types of media with three types of explants: cotyledons, hypocotyls, and roots. More than 93% of cultured explants from both species could form calli or calli with shoots. With regard to shoot formation, A. canadensis could produce multiple shoots from all types of explants more efficiently than A. racemosus. The highest shoot induction was approximately three shoots per explant in A. racemosus, whereas A. canadensis could reach ≈10 shoots per explant. M1 could induce more shoots than M2 no matter what type of explant was used, but the overall induction rates were no significant difference. Among the three types of explants used, the cotyledons were the best explants for shoot induction in A. canadensis, whereas hypocotyls were the best in A. racemosus. In A. racemosus, shoots could also be obtained from calli on the rooting medium containing Murashige and Skoog basal plus vitamins, 2.84 μm indole-3 acetic acid, 2.5% (w·v−1) sucrose, and 0.7% (w·v−1) agar (pH 5.7). Approximately 43% of A. canadensis shoots and 19% of A. racemosus shoots could be rooted on the rooting medium.