Abstract: 【Objective】A Long-term in vitro culture plant regeneration system of Petunia hybrida is useful not only in obtaining somatic variants and thereafter to select superior lines, but also in analyzing the stability of foreign gene in the cells during long-term culture and plant regeneration. 【Method】Different callus were obtained by culturing the leave explants of one double flower Petunia plant on the media supplemented with different combinations of auxin and cytokinnins. Calli were sub-cultured on the media of MS+ BA 2.0 mg•L-1+0.5 mg•L-1 NAA+200 mg•L-1 CH and MS+ BA 0.5 mg•L-1+0.5 mg•L-1 NAA+200 mg•L-1 CH for more than 3 years under different light condition. The total DNA of 20 regenerated plants were analyzed by ISSR utilizing 8 primers. 【Result】 Plants were successfully regenerated from Long-term cultured callus when transferred onto shoot induction media supplemented with low concentration of PGR. After continuous culture on the media for at least three times, all adventitious shoots developed into complete plants of normal morphological characteristics. ISSR analysis showed that there was dramatic genetic variation among somatic variants. 【Conclusion】Plant regeneration could be successfully implemented after long-term subculture of the callus of Petunia hybrida in higher concentration BA media when they were transferred onto shoot induction media supplemented with low concentration of PGR. The culture system presented here is effective in obtaining somatic variants for Petunia hybrida.