<p>Ethiopian potato is an underutilized tuber crop with cultural and medicinal values, cultivated by smallholder farmers mainly for food. The study was conducted to develop an efficient protocol for generating callus, somatic embryo, and in vitro plant regeneration in three genotypes of Ethiopian potato. A combination of 2,4-dichlorophenoxyacetic acid (2,4-D) and Indole 3 acetic acid (IAA) with 6-benzylaminopurine (BAP) and kinetin (Kn) resulted in more efficient callus induction than 2,4-D alone. None of the three genotypes showed callus induction in Murashige and Skoog (MS) media devoid of plant hormones. The two auxins (2,4-D and IAA) in combination with Kn were effective in promoting embryogenic callus development in all tested genotypes. The highest mean number of somatic embryos was found in genotype Unnuka, followed by Lofuwa in half-strength MS containing 0.2 mgL^− 1 Kn + 0.1 mgL^− 1 IAA, whereas genotype Chankua had the highest mean number of somatic embryos in half-strength MS containing 0.4 mgL^− 1 Kn + 0.1 mgL^− 1 IAA. Unnuka had the highest shoot regeneration frequency, number of shoots, and shoots with roots in 1 mgL^− 1 BAP + 0.5 mgL^− 1 gibberellic acid (GA₃). Lofuwa and Chankua, however, showed the highest results with 0.5 mgL^− 1 thidiazuron (TDZ) + 0.5 mgL^− 1 GA₃. Plantlets survived at a frequency of 92%, 88%, and 80% for Chankua, Unnuka, and Lofuwa, respectively, on a substrate mixture of river sand and soil in a 2:1 ratio. In this study, the type and optimum concentration of plant growth regulators required for somatic embryogenesis were determined for the three genotypes of Ethiopian potato.</p>