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  determine sequence variation and identify SNPs in MIC-3 family members of selected diploid and tetraploid species in cotton;  identify haplotypes associated with these MIC-3 family members and  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.