ABSTRACT Three Gossypium species have been used to breed cotton as they vary in their fiber production and resistance to stresses. Transcription factors (TFs) mostly are present in different copies or isoforms by which they conduct their regulation. Their copy number can determine organism behavior to a cue. Simple sequence repeats (SSRs) are one of the most informative and versatile molecular markers. Transcription factors of three Gossypium species were compared in silico. Seventy eight percent of TFs were common between the three species. Single copy for each species were 6057 TF. Gossypium hirsutum and G. raimondii shared the most common interspecific TF. Gossypium arboreum species-specific TF were the least. MYB TF family with its subfamilies is the most abundant followed by bHLH and AP2/ERF family. Gossypium hirsutum generally possesses more TF copies compared to other two species. The 2109 single-copy clusters indicate that G. hirsutum has received one copy from only one parent. The five most abundant SSR markers of TF were dinucleotides AT, TA, TC, CT and TG belonging to G. raimondii. For G. arboreum and G. hirsutum they were trinucleotides CAA, CGA, TGA, GAA (CAT: G. hirsutum) and TCA. The findings suggest that there is regulatory difference between the three Gossypium species for fiber production and insect attack response. The differences may be due to some adaptive deletion events during speciation of G. hirsutum from its parents G. arboreum and G. raimondii.