MIC-3 is a recently identified gene shown to exhibit increased root-specific expression following nematode infection of plants that are resistant to root-knot nematode (RKN).� We cloned and sequenced PCR-amplicons (derived from MIC-3-specific degenerate primers) from individual plant DNAs to [1] determine sequence variation and identify SNPs in MIC-3 family members of selected diploid and tetraploid species in cotton; [2] identify haplotypes associated with these MIC-3 family members and [3] discover the chromosomal location(s) of the MIC-3 family members.� SNP markers were identified from MIC-3 genomic sequences of four tetraploid species (Gossypium hirsutum, G. barbadense, G. tomentosum, G. mustelinum) and three diploid species (G. herbacium, G. raimondii and G. longicalyx).� Phylogenetic trees were constructed based on the most parsimonious algorithm of the PAUP software.� Comparative analysis of tetraploid sequences within individual clade of the Phylogram suggested several putative haplotype variants.� Five different SNP markers, associated with five different MIC-3 family members, were delimited to chromosome arm 04sh by hypoaneuploid cytogenetic deficiency tests.� Intergenic regions of these clustered genes were similarly located on the same chromosome arm. �Multiple sequence alignments of consensus sequences of MIC-3 family members showed high level of homology with the GenBank EST sequences, isolated from the Fusarium wilt and RKN infected cotton roots.� Results suggested that some members of MIC-3 gene family were associated with the response to RKN and Fusarium oxysporum f. sp. vasinfectum infection.� It has been reported previously that RKN increases the incidence and severity of cotton to Fusarium wilt.� The findings document a strategy for developing SNP markers for candidate genes in cotton, demonstrate the clustered organization of various MIC-3 family members, and provide molecular evidence on the association of a candidate gene with both Fusarium wilt and RKN diseases in cotton.