Tanacetum balsamita L. (Costmary) has long history of use as an important medicinal herb for treatment of many diseases. The objective of the present study was to establish an in-vitro tissue culture protocol for calli production and somaclonal variation study of calli derived from different plant parts of costmary. Nodal segments, petiole and leaf disc explants were employed for calli production on 6 different MS media supplemented with different combinations of 2,4-D (1 and 2 mg/L) and BAP (0.2, 0.5 and 1 mg/L) and two magnesium ion concentrations (185 and 370 mg/L). Callus production was significantly higher on MgMS basal medium containing 1 mg/L 2,4-D + 1 mg/L BAP. To assess somaclonal variation, genomic DNA was extracted from calli of fourth subculture. Ten RAPD primers were used to analyze genetic variation between callus samples. The results showed different amplification patterns between callus samples from different subcultures.