An important feature of A. rhizogenes-induced hairy roots is their unique ability for investigation of gene function and production of secondary metabolites such as diosgenin in fenugreek. In order to evaluate the transformation frequency and the efficiency of transgenic hairy root induction, leaf and stem explants from two fenugreek ecotypes, Karaj and Bushehr, were infected with three concentrations of $OD_{600}=0.8$, 1.2, and 1.6 of A. rhizogenes strain K599 harboring a GFP gene. Regardless of ecotype, the ability of stem explants for the induction of hairy roots (8.09) and the transformation frequency (81.3%) was higher compared with leaf explants with the values of 5.97 and 71.88%, respectively. The number of transgenic GFP-positive hairy roots ranged from 4.2 to 13.5 in the Karaj ecotype and 3.8 to 9.9 in Bushehr. The highest transgenic hairy root (8.76), the transformation frequency (79.76%), and the growth rate of transgenic roots ($0.77d^{-1}$) were obtained from infection with K599 at $OD_{600}=1.2$, while the lowest belonged to the bacterial concentration of $OD_{600}=1.6$. Although the ecotype Bushehr had lower total roots (7.53) and transgenic hairy roots (6.08), it showed higher transformation frequency (79.56%) than Karaj (73.63%). Therefore, the results indicate the importance of genotype, type of explant and bacterial concentration in breeding for induction of transgenic hairy roots and consequently, production of secondary metabolites in fenugreek.