<p>Breeders normally select genotypes based on morphological characters, primarily regulated by nuclear genome. However, cytoplasmic genome, including mitochondria and chloroplast genomes, plays an important role to perform a number of biological functions in co on breeding. e characteristics and the mode of genetic inheritance of cytoplasm and nuclear genome provide complementary valuable information to study gene ow, evolution, and population history. e study is aimed to develop cytoplasmic DNA markers as a tool for genetic research and to use the DNA markers as a tool to detect genetic variation in the cytoplasmic and nuclear genome in a set of G. hirsutum and G. barbadense lines. e study involved 61 SSR primer pairs associated with important ber speci c traits of the nuclear genome and 49 indel and SSR primers speci c to the cytoplasmic genome to screen 20 G. hirsutum and 74 G. barbadense lines om diverse geographical locations. It showed that cytoplasmic indel markers are more polymorphic compared to the cytoplasmic SSR markers among the lines. e overall results showed that the selected 94 lines could widely be divided into two broad groups of G. hirsutum and G. barbadense. e research provided for the rst time a genetic tool to study co on cytoplasmic genome and a report comparing the genetic diversity in the cytoplasmic and nuclear genomes of G. barbadense and G. hirsutum lines. e following research will help breeders to develop a breeding strategy maximizing the e ects of genetic diversity to improve co on lines.</p>