Abstract: This research describes the optimized method of carrot hairy roots production for monoxenic culture of Arbuscular Mycorrhizal Fungi (AMF) in Iran. Inoculation of mature carrot root sections was carried out using four isolates of Agrobacterium rhizogenes as well as a mixture of isolates that had been grown in three culture media (LB, YMA and modified medium) with different incubation times (24 and 36 h) before inoculation. Three methods of carrot hairy root initiation were compared in an attempt to optimize hairy root production in terms of the number of root initiations and the health (lack of rotting) of the explants. Results showed that most treatments resulted in hairy root formation, although not equally so. It was shown that the mixture of bacterial strains from three culture media increased the frequency of bacterial colonies with a good transformation characteristic. Also, incubation of inoculated carrot discs for 48-72h in darkness provided a suitable condition for bacterial strains to insert their copies of Ri T-DNA. The source of the carrots was one of the most important factors. With mature, freshly harvested carrots showing better hairy root production. Dissection of the cortex to expose the cambium had a positive effect on hairy root initiation. Increasing bacterial strains incubation time from 24 to 36 h improved transformation efficiency in all treatments but not in method III. Results showed that the best method for initiation of hairy root induction in carrots especially in Iran is the method I.