Recently the swift development of methods for a rapid efficient synthesis of synthetic oligonucleotides with the discovery of polymerase chain reaction (PCR) provides a rapid, safe and efficient way to develop a genome mapping program. However, the current trend in PCR in plant genome research is primarily based on RAPD markers. Here, we report an alternative approach of RAPD to develop a genetic map of molecular markers in Gossypium species utilizing amplification of specific DNA sequences of single or low copy genes or genes of biological interest. This specific sequence based DNA markers have several advantages: 1) it overcomes the need for an expensive, long term, centralized repository of clones because the information related to the sequence and the marker can be stored in a database; 2) it will improve the quality control and project accountability because mapping results can be checked by a scientist using the information from the database and 3) it will subsequently help in cloning genes of biological importance that can be beneficial in future for genetic manipulation. Cotton genoic DNAs were amplified with conserved flanking sequences of heat shock proteins in Pisum sativum, homeobox genes of Drosophilia melanogastor and alpha amylase gene in Vigna mungo. As part of the initial effort to develop genomic map of cotton we would like to report the chromosomal assignment of three molecular markers in cotton Interspecific crosses of G. hirsutum and G. barbadense and monosomic, monotelodisomic cytogenetic stocks and substitution lines in G. hirsutum were used to determine the chromosomal location of the markers. Synthetic oligonucleotides complementary to two 24 mer primers (3' and 5' end) of heat sock proteins in Pisum sativum and two 18 mer primers (3' and 5' end) of alpha amylase gene in Vigna mungo were synthesized. Genomic DNA of Fl monosomic interspecific chromosome substitution line was used for amplification in a polymerase chain reaction (PCR) with the synthetic primers. The results revealed the presence of seven amplified bands with the heat shock protein ranging from 200 to 500 bp, and nine amplified bands with the alpha amylase gene ranging from 200 to 1500 bp. Our preliminary results from the segregation analysis of monosomic Fl plants in PCR suggested that the amplified band of 450 bp with heat shock protein is on chromosome 10 and the amplified bands of 1200 bp and 1500 bp of alpha amylase gene are located on chromosome 12. The homeobox gene failed to reveal polumorphism at the interspefic level of tetraplaid coton.