Colchicine has been used to induce chromosome doubling in plant species. Diploid cotton species have inherited desirable genes that are resistant to biotic and abiotic stresses and also possesses useful fiber quality traits, such as higher fiber length and strength. To transfer these desirable traits to the most extensively cultivated tetraploid cotton species through hybridization, it is a prerequisite to double the chromosome number of the diploid species. In this study we used Gossypium herbaceum L., Gossypium arboreum L. and Gossypium raimondii L applied two experiments: seedling, and embryo culture treatments. The results revealed that colchicine treatment was more effective in seedling treatment when the length of hypocotyls ranged from 5 to 8 mm. Further, we observed the optimum treated in the three species as the concentration of colchicine 0.5% and duration of treatment 8 & 16 hrs in seedling experiment while in embryo culture experiment the concentration of colchicines 2000ppm duration 24 hrs treatment its response. Among the experiments employed to induce polyploidy in the diploid cotton species, seedling experiment was the most effective and consistent as compared to embryo culture experiment. Though embryo culture demands less experimental space and little amount of colchicine, however the high risk of contamination and high sensitivity of embryo to colchicine make it less preferable. Colchicine treated plants are susceptible to environmental stimuli induced by high temperature and should be kept away from direct sunlight, the optimum dose of colchicine and the incubation time depends on the species and the environmental conditions.