The ability of high-frequency regeneration is the key factor for successful chloroplast transformation of rice. At the present study, twenty newly bred rice lines with early-maturing and good agronomic traits were used as explants to evaluate their regeneration ability by means of optimal design with three media and 20 lines, and finally screen out the lines with high-frequency regeneration ability. The results indicated that the callus induction frequency of line 19 reached up to 100% on the MS medium supplemented with 2.0mg/L 2,4-D, and the callus differentiation rate kept 77.8% on SF3 medium after eight rounds of subculture continuously. More importantly, high-frequency differentiation rate of line 19 which means more than 10 plantlets were regenerated from one callus still remained 36.1%, and therefore it is considered to be the potential candidate for subsequently chloroplast transformation.