Cotton is one of the most important crop worldwide and in India. Genetically modified cotton crop was officially introduced in the year 2002. Genetically modified crop varieties have various advantages over traditional varieties. This cotton carried genes from the bacterium Bacillus thuringiensis (Bt), therefore it is called as Bt cotton. Bacillus thuringiensis is a gram-positive, spore-forming bacteria, generates a number of insecticidal crystal proteins (ICPs) that are poisonous to larvae of lepidopterans, dipterans, and coleopterans. In recent years, transgenic Bt cotton varieties have been widely grown, which effectively controls cotton bollworm and reduce the application of insecticides. Continuous cultivation of Bt-cotton is known to have negative impact on soil microflora. It has been reported that Bt toxin leach out in the rhizosphere and inhibits the growth of soil microbes. Therefore, the present study was undertaken to study the impact of Bt cotton and non-Bt cotton cultivation on soil microflora at different stages of cotton plants in Diksai, Jalgoan and Maharashtra. The obtained results revealed that Bt-cotton crop adversely affected the below ground biodiversity of microbial population.