Following the sequencing of rice genome, the functional analysis of unidentified genes is gaining wide importance. For these, mutant isolation is one of the effective ways to isolate and clone the target genes and analyze their functions. For finding the various mutants in the same genetic background, seeds of Oryza sativa cv. Nipponbare were treated with ethyl methane sulphonate (EMS). A total of 1056 mutants for five categories were screened in M2 generation with the seedling frequency of 26.29‰ at three-leaf stage, but only 264 mutants were verified in M3 generation with a frequency of 6.57‰. Among the mutants verified in M3 generation, the frequency of leaf mutation was highest (2.22‰), followed by seedling height (1.74‰) and abiotic stress tolerance mutant (1.47‰). Nineteen characteristic mutations, including a big group of abiotic stress tolerant mutants such as herbicide resistant, salt tolerance and drought tolerance were identified at this stage. By observation of rice growth characteristics at different developmental stages, another 220 mutants have been isolated and verified in the M3 generation with the mutant frequency of 53.9‰ covering about 28 mutant taits. Among those identified, the highest frequency were obtained for appearance of brown rice mutant with 18.37‰ followed by panicle mutant with 13.47‰ and grain mutants with 9.06‰, respectively. All the mutants screened above were suitable for gene function analysis and for utilization in agronomy.