Mutants are excellent genetic materials for revealing molecular mechanisms of important genes. In this research, the cotton seeds were mutagenized by different concentrations of EMS solution (0%, 0.2%, 0.5%, 1.0% and 1.5%) and processing duration (4 h, 8 h, 10 h and 12 h), respectively, in order to optimize the reaction condition for EMS mutagenesis in upland cotton. Then, the effect of different EMS concentrations and treatment duration on germination rate and growth of sprouted cotton seeds was analyzed and the morphological characters of M2 and M3 generation were identified. The results showed that 1.0% EMS for 10 h was optimal for Lu6269 mutagenesis. The seed germination rate was about 49%, and the half lethal concentration is 1.0%. To obtain abundant mutants, approximated 10000 seeds of Lu6269 were treated with 1.0% EMS for 10h. A fiber abnormal mutant (the fiber non-fluffy in the development of maturation, sfm-1) was found in the M2 generation, and this phenotype was inherited by observing M3 generation seeds. This mutant showed a large cavity between the embryo and the seed coat, a weak and small embryo as well as decreased unearthing of seeds. Taken together, by establishing the EMS mutagenesis system of G. hirsutum cv Lu6269, we obtained sfm-1, a stable mutant for abnormal seed and fiber development, which served as the material foundation for fiber development, seed germination and function genome research in cotton.