Melissa officinalis L., due to its useful application in medicine, is being paid more attention. In order to establish a stable regeneration system with 4 landraces collected from different climate in Iran, major parameters such as regeneration rate, rooting percentage, shooting induction, proliferation rate, fresh and dry weight as a biomass of cells were investigated. Statistical analysis of results showed that BAP in combination with NAA had the highest regeneration in shoot tips explants. NAA in combination with IAA and kinetin had the best response to callus induction. Also 1 mgl/l NAA had a higher response to rooting than other auxins used. 2,4-D at 1.0 mg/l and BAP at 0.5 mg/l showed the highest production of fresh and dry weight, 5.48 and 0.407 g, respectively, that is approximately 20 times the initial weight of callus. 2,4-D (1 mg/l) and BAP (0.5 mg/l) had the highest cells number.