Cotton fiber is the most important raw material used in textile industry throughout the world. Due to demand of good quality fiber for manufacture of fabrics, there is need to develop cotton varieties with improved fiber characteristics. Through conventional breeding, a number of cotton varieties have been developed. However, this technique has disadvantage of narrow germplasm and requirement of longer time period for selection of best lines. Using genetic engineering techniques, fiber genes may be directly introduced in cotton which may express under the control of certain promoters. Through gene expression profiles, tissue and developmental stage specific expression level of genes can be determined. Gene those have been expressed specifically in fibers or other tissues may be transformed to alter various characters. Upstream regulatory regions of theses gene may also be evaluated as promoter. Fully characterized promoters can be used as component of expression cassette for crop improvement through molecular breeding. Cotton fibers are unicellular seed trichomes and the largest known plant cells. Fiber development in cotton is a complex process involving a large number of genes expressed during each fiber development stage. The objective of the study was to determine expression level of fiber related genes in cotton. The expression level of eight fiber related genes (EXPANSIN, tubulins, LTP1, LTP3, LTP7, E6, SPS, and susy) was determined in cotton tissues using reverse transcriptase real time PCR. Three cotton (Gossypium hirsutum) lines of varying fiber length were used to study the effect of genotypes on gene expression. The results revealed that transcription level of most of genes elevated from 5 to 20 DPA fibers. All genes showed similar expression pattern in all three genotypes. However, EXPANSIN was found to be highly expressed in long fiber variety during rapid elongation period than in medium and short fiber length. Sucrose synthase showed high expression in 15-20 DPA fibers and roots. Expression data may help also to explore the role of particular gene in fiber development. On the basis of expression profiles, two genes (SPS and LTP7) were selected. Upstream sequences of both genes were retrieved through high through put genomic sequences (HTGS) and analyzed using bioinformatics tool. About 2 kb region upstream of SPS and 1.8 kb upstream of LTP7 gene were selected for promoter isolation. Patent BLAST results of SPS and LTP7 promoter sequences confirmed that both were novel regulatory regions. Analysis of both promoter sequences using plantCARE revealed basal promoter elements including TATA box and CAAT box. There were various light regulated motifs, MYB binding sites and stress related motifs. Various motifs related to transcription activation and tissue specific expression were also identified in both promoters. Analysis of promoter sequences using plantPAN identified binding sites for transcription factors including MYBS, ERELEE4, GAREAT, ABRELATERD1 and WBOXNTERF3. Both SPS and LTP7 promoters along with their deletion fragments were isolated from genomic DNA of cotton leaves. A 1.5 kb deletion fragment for SPS promoter was cloned in expression vector. Two 5′ deletion fragments for LTP7 promoter (1 kb and 1.5 kb) were also cloned. Transient GUS expression assay revealed that full length SPS and LTP7 promoters were active in fiber tissues. Deletion derivatives (1.5 kb) of both promoters showed relatively weak GUS expression in fiber tissues. However, 1 kb LTP7 promoter was highly active in fiber tissues. No GUS expression was detected in root, shoot and leaf tissues. Agrobacterium mediated transformation in tobacco revealed that SPS promoter exhibited GUS stain in tobacco leaves in the form of patches while its 1.5 kb deletion fragment showed much low expression. Full length LTP7 promoter along with 1 kb LTP7 showed high vascular tissues specific expression in tobacco leaves. The novel promoters may be used to derive fiber specific expression in cotton and vascular tissues specific expression in heterologous system. The indigenous promoters can be used for specific expression of transgenes without IPR issues.