Justicidin B, an arylnaphthalene lignan, has strong cytotoxicity on chronic myeloid and chronic lymphoid leukemia cell lines. The first report of the production of justicidin B in a Linum species concerned in vitro culture of Linum austriacum. Therefore, culture characterization and presence of arylnaphthalene-type lignans in calli and plantlets of Linum tenuifolium from section Linastrum, Linum bienne, and Linum glaucum from section Linum were studied. Seed germination of L. tenuifolium in the light and darkness was significantly higher (p < 0.05) than of L. bienne in the light and L. glaucum in the darkness. L. tenuifolium seedling length in the darkness was significantly higher (p < 0.01) than under light conditions. There were no significant differences in the calli and shoot biomass weight, number and length of shoots in three species over one month, while the shoot diameter of L. bienne was significantly different (p < 0.05) from that of L. glaucum. Justicidin B was detected in L. glaucum callus and plantlet cultures by HPLC/MS/UV-DAD and HPLC coupled with a photodiode array detector. This finding is important from pharmaceutical point of view and shows the chemosystematic relation between L. glaucum and L. austriacum and this method will be a powerful tool for detecting natural products in interested and endangered medicinal plants.