Diseases cause significant losses in cotton production throughout the US Cotton Belt. Growing resistant cultivars can significantly improve cotton yields and effectively reduce production inputs. Disease resistance (R) genes have been isolated in numerous plant species and the R genes with domains of nucleotide binding sites (NB) and leucine rich repeats (LRR) represent the largest R gene family. Degenerate primers designed based on conserved motifs of plant disease resistance genes were used alone or in combination with AFLP primers to analyze disease resistance gene analogs (RGAs) in a recombinant inbred line (RIL) population of Pima (Gossypium barbadense) 3-79 and Upland cotton (G. hirsutum) line NM 24016. Eighty-eight polymorphic RGA markers were amplified by 8 pairs of RGA degenerate primers, while 131 polymorphic RGA-AFLP markers were produced from six pairs of RGA-AFLP primer combinations. Of the 219 polymorphic RGA and RGA-AFLP markers that were identified, 212 were assigned to 18 chromosomes and linkage groups based on existing SSR markers that are on known chromosomes. However, the RGA and RGA-AFLP markers are not evenly distributed among chromosomes in that 189 RGA and RGA-AFLP markers (88%) are assigned onto three ''giant'' chromosomes, i.e., C6, C12, and C15, suggesting RGA clusters in the cotton genome. Several RGA and RGA-AFLP markers were mapped to the same linkage group carrying a root-knot nematode resistance gene. The identification and mapping of RGA and RGA-AFLP markers provide a framework to facilitate marker-assisted selection of disease resistance in cotton breeding and to understand the physical relationship of cotton resistance genes.