Silybum marianum L. from the Asteraceae is an annual or biennial plant widely distributed in the north, west and south of Iran. The fruits are containing medicinal flavonoids such as silymarin. This study was conducted to optimize the growing conditions for hairy roots and to assay the diverse factors affecting the induction and growth of hairy roots induced by Agrobacterium rhizogenes. The seeds were cultured on MS medium and were kept in the growth chamber. After germination and seedling growth; explants from the leaves, hypocotyl and roots were taken behind growth and after 5, 10 and 15 days. The explants were inoculated (treated) with two (R1000 and 15834) Agrobacterium rhizogenes strains. Furthermore, the effects of two inoculation methods (floating and injection) were assayed on the hairy roots emergence percentage. MS and 1/2 MS growing media were compared regarding the establishment of the hairy roots in the liquid medium. In a separate experiment, the effect of sucrose amount (20, 30 and 40 gl^-1) were evaluated on hairy root induction and the roots emergence percentage was recorded. To verify the transgenic identity, PCR test with specific marker genes was carried out. The results revealed that 15834 strain was the strain of choice in 5 days old hypocotyl explants and produced more hairy roots than other strain. The best rooting percentage belonged to leaf explants at 15 days-old, treated with R1000. Floating was the method of choice for inoculation. 30 gL^-1 sucrose was the best treatment for the root induction as well.