Technical Abstract: New source of molecular markers accelerates the efforts in improving cotton fiber traits and aid in developing high-density integrated genetic maps. We developed new markers based on candidate genes and G. arboreum expressed sequence tag (EST) sequences, and validated them through amplification, genetic and physical mapping. Nineteen gene-based markers were successfully amplified across five tetraploid and 21 diploid Gossypium species. Cluster analysis of fragment data generated a phylogenetic tree with four major sub-clusters for 23 species while three species branched out individually. A higher level of polymorphism (~58%) was observed through the use of cleaved amplified fragment polymorphism (CAP) techniques between G. hirstutum and G. barbadense. Euploid G. barbadense chromosome substitution (CS-B) lines were employed to locate the 11 CAPs markers of which five CAPs markers were localized to seven different chromosomes in the cotton genome. Two hundred A-genome based simple sequence repeat (SSR) markers were designed after data mining of G. arboreum ESTs (Mississippi Gossypium arboreum EST-SSR – MGAES). Over 70% of MGAES markers successfully produced amplicons while 65 of them demonstrated polymorphism between the parents of G. hirsutum and G. barbadense recombinant inbred line (RIL) population. When used to determine linkage relationships among them, MGAES markers formed 14 linkage groups that were assigned to 11 chromosomes and 11 chromosome arms in cotton genome through the use of euploid/hypoaneuploid CS-B analysis. Gene-based and MGAES markers were highly informative as they were designed from candidate genes and fiber transcriptome with a potential to be integrated into the existing cotton genetic and physical maps.