To supply high-quality cotton fibre for the textile industry, the development of long, strong and fine fibre cotton varieties is imperative. An interlinked approach was used to comprehend the role of fibre genes by analyzing interspecific progenies of cotton species. Wild Gossypium species and races are rich source of genetic polymorphism due to environmental dispersal and continuous natural selection. These genetic resources hold mass of outclass genes that can be used in cotton improvement breeding programs to exploit possible traits such as fibre quality, abiotic stress tolerance, and disease and insect resistance. Therefore, use of new molecular techniques such as genomics, transcriptomics and bioinformatics is very important to utilize the genetic potential of wild species in cotton improvement programs. Interspecific lines and Gossypium species used in the study were grown at Central Cotton Research Institute (CCRI), Multan. After retrieving DNA sequence of the genes from NCBI, the primers for gene expression and full-length gene sequence were designed. Expression profiling of Expansin A4, BURP Domain protein RD22-like and E6-like fibre genes was performed through Real Time PCR. BLAST and DNA sequence alignment was conducted for sequence comparison of interspecific lines and Gossypium species. Different in silico analysis were used for characterization of fibre genes and identification of cis acting promoter elements in promoter region. Variable expression of genes related to fibre development was observed at different stages. BLAST and DNA sequence alignment demonstrated resemblance of interspecific lines with G. hirsutum. In silico analysis on the sequence data also confirmed the role of Expansin A4, BURP Domain protein RD22-like and E6-like fibre genes in fibre development. Genetic engineering is also recommended by transferring E6-like, Expansin A4 and BURP Domain RD22-like genes in local cotton cultivars. Similarly, several stress tolerant and light responsive cis acting elements were identified through promotor analysis, which may contribute for fibre development in the breeding programs. Expansin A4, BURP Domain RD22-like and E6-like have positive role in fibre development with variable expression at fiber length and strength associated stages.