We used an integrated approach of PCR and RFLP in developing molecular markers in cotton. PCR was used to amplify specific DNA sequences of singly and low copy genes of biological importance in the cotton genome. The amplified products were isolated and used as probes in an RFLP mapping program. This strategy of genome mapping will not only provide the molecular genetic information of single and low copy genes, but also can be beneficial in cloning genes of biological importance in cotton genome. Inter and Intraspecific crosses between Gossypium hirsutum and G. barbadense as well as monosomic, monotelodisomic cytogenetic stocks and chromosome substitution lines were used to determine the chromosomal location of the molecular markers. Primers were designed to amplify specific regions of known gene sequences. Twenty 20-25-mer synthetic oligonucleotides complementary to the 5' and 3' ends of DNA sequences of single and low copy genes were synthesized. We have developed a suitable DNA extraction method in cotton without using the tedious and hazardous cesium chloride gradient technique. Cotton contains a high level of secondary products that interfere in extracting clean DNA. The primers were amplified for 40 cycles at 94_C for 30 sec, 60_C for 1 min., 72_C for 2 min. and 72_C for 10 min for 1 cycle. Our initial results indicated that many of these sequences are conserved across the species. We isolated several probes from the amplified products including two DNA products of 320 bp and 500 bp from a specific fiber quality protein gene as well as a 750 bp product from a copia-like retrotransposable element. We have developed a suitable RFLP procedure for cotton genome mapping using these probes. Our results revealed the existence of polymorphisms for the Acc synthase gene (Bam HI), fiber gene (Hind III) and the copia-like element (Hind III). We are in the process of screening monosomic F1 plants with our combined protocol of PCR and RFLP to identify the chromosomal location of these molecular markers. This strategy will not only provide the chromosomal location of the molecular markers, but also will eventually provide sequence information for the specific region of the chromosome that can be used subsequently in the chromosome walking technique for further genome analysis.