A diversity array technology (DArT) marker platform was developed for the cotton genome, to evaluate the use of DArT markers compared with AFLP markers in mapping and transferability across the mapping populations. We used a reference genetic map of tetraploid Gossypium L. that already contained ~5000 loci, which coalesced into 26 chromosomes, to anchor newly developed DArT and AFLP markers with the aim of further improving utility and map resolution. Our results indicated that the percentage of polymorphic DArT markers that could be genetically mapped (78.15%) was much higher than that of AFLP markers (22.28%). Sequence analysis of DArT markers indicated that a majority matched known expressed sequence tag (EST) sequences from tetraploid and diploid Gossypium species. A total of 794 Arabidopsis genes were homologous with various DArT marker sequences. Chromosomes 5(A), 7(A), 19(D), 23(D), and 24(D) had more Arabidopsis syntenic DArT markers than the other chromosomes. Anchoring DArT markers from the reference map to a recombinant inbred line (RIL) map indicated that DArT markers will speed the building of maps in de novo RIL populations.