Introduction: Tissue culture is a vital tool in plant biotechnology, enabling the rapid propagation, genetic modification, and breeding of valuable crop species. In Cannabis sativa L., understanding and developing effective tissue culture protocols is important due to the plant's high demand, the need for pathogen free propagation, the potential to speed up breeding programs, and the race to understand the plant’s biology. This project focused on establishing protocols and analysis of seedling germination of a wide range of cultivars of C. sativa in vitro, callus culture from cotyledons, hypocotyls, and other organs, and indirect organogenesis from callus. Data were analyzed to make recommendations for future researchers looking for cultivars that perform well in aseptic conditions. A total of 37 accessions were introduced to tissue culture as seeds. Of those, 34 were also germinated in greenhouse conditions. Thirty varieties successfully germinated in vitro, and explants were transferred to callus induction media. Of those, 21 had data collected on callus development. All varieties produced callus, including green callus. Callus from each were transferred to shoot induction media, but data were not collected on shoot induction because shoots did not develop from hypocotyl or cotyledon explants.