Biochemical and molecular markers have proven to be powerful tools for discerning bio-systematic, bio-geographic, and phylogenetic relationships. Bio-systematic information can be important for guiding traditional breeding programs, gene transfer, interspecific hybridization, and gene conservation. Polymorphism is a result of variation in the genome of an individual plant or animal organism. The present investigation of an experiment is conducted to explore the magnitude of polymorphism in cotton genotypes using biochemical and molecular marker techniques. In the present investigation, the cotton lines exhibited polymorphism is analyzed based on the relative mobility of bands. The relative mobility (Rm) values ranged higher in protein banding pattern followed by carboxylase esterases, peroxidases, and polyphenol oxidase activity studied by SDS-PAGE and PAGE electrophoresis techniques. In respect of the molecular investigation using RAPD, the primers used exhibited satisfactory amplification essential for discriminating the population. The most amplified primers exhibited an average polymorphism of about 73 percent. The biochemical and molecular marker techniques are found to be quicker for the screening of a large gene pool and clustering the genotypes into different diverse clusters which are useful to select the parents for hybridization. These techniques can assist plant botanists especially involved in plant breeding activities to speed up the conventional cotton improvement program. Further, it can be concluded that the biochemical and molecular markers investigated in the present experiment are adequate to judge the dissimilarity among the genotypes for speeding up the selection of parents with assumption that the banding profile is a measure of discrimination.