Abstract: In order to solve the shortage of virus-free seed yam production, with its stem section as the test materials, the ratio of different plant growth regulators on the impact of ‘Qi’ yam in vitro shoot regeneration as well as the influence of different carbon sources on the in vitro micro tuber formation were studied. The ‘Qi’ yam efficient in vitro shoot regeneration system and in vitro production method of micro tubers were established. The results showed that it was useful for ‘Qi’ yam adventitious bud regeneration in the MS media with 0.5 mg/L 6-BA and 0.1 mg/L NAA or with 0.2 mg/L ZT and 0.2 mg/L NAA, and the ratio of ‘Qi’ yam adventitious bud differentiation reached 81.33% and 82.67%, respectively. In the MS media with 0.2 mg/L 6-BA and 0.02 mg/L NAA, ‘Qi’ yam adventitious bud multiplication was effective and multiplication factor was as high as 4.7. The MS media with 0.02 mg/L NAA was suitable for the root growth of the ‘Qi’ yam plants, and the rooting rate was reached 96.32%. 150 mg/L PVP could effectively reduced the brown phenomenon in the ‘Qi’ yam tissue culture. The production method of in vitro micro tubers of Dioscorea opposita ‘Qi’ has also been established. The most favorable formation rate of ‘Qi’ yam micro tuber was gained in the MS media with 0.5 mg/L 6-BA, 0.05 mg/L NAA and 60 g/L sucrose. In these conditions, the highest micro tuber formation rate was 97.50%, and the micro tuber weight reached 0.18 g and 0.39 g after 60 days and 120 days, respectively.