Effective long-term conservation of rice germplasm resources is crucial to world food security, and cryopreservation is the best way to achieve this goal. In this study, a method with rice embryogenic callus high sugar preculture and direct liquid nitrogen cryopreservation was established, and the callus regeneration rate and the differentiation rate of regenerated calli were used to evaluate the cryopreservation efficiency directly. The results showed that the callus regeneration rates of japonica rice ‘Nipponbare’ and indica rice ‘Hanhui 3’ with sizes of 1.6-3.0 mm and >3.0mm was significantly higher than those of <1.6mm after cryopreservation. The callus regeneration rates of ‘Hanhui 3’ after preculture with 100 g/L sucrose and 170 g/L sucrose were significantly higher than those after preculture with 240 g/L sucrose. The callus regeneration rates of japonica rice ‘WDR48’, japonica rice ‘Nihonbare’ and indica rice ‘Hanhui 3’ treated only with high sugar preculture were significantly higher than those treated with vitrification. Simple Sequence Length Polymorphism (SSLP) identification indicated that seedlings regenerated from calli either with or without cryopreservation were genetically stable. The study indicated that callus preculture with high glucose and direct cryopreservation is an easy, high-throughput, stable and reliable method for long-term preservation of rice germplasm resources.