Background: Cotton fibers, derived from the seed coat and used in the global textile industry for fabric production, are generally recognized as the predominant form of individual unicellular cells. The investigation of cellular proliferation and differentiation may be efficiently conducted by using cotton fiber production as a model system. Expression profiling techniques are very helpful to determine the tissue (stem and roots) and stage (0, 05, 10, 15 and 20 DPA) specific gene expression in cotton fiber. This profiling technique is helpful in the development of a new plant (variety) through transformation, resulting in the development of a new plant with desirable fiber characteristics. Methods: The expression profiling was carried out in upland cotton genotypes with variable staple length including: i) long staple length, ii) medium staple length and iii) short staple length) at five different days post anthesis (DPA) phases of fiber elongation (0, 5, 10, 15 and 20 DPA) through real-time PCR. Two internal controls (ubiquitin and 18sr RNA) genes were used for data normalization. Current research has focused on genetic basis of fiber regulation and understanding the molecular basis of fiber development in upland cotton (G. hirsutum L.) genotypes (‘CYTO-179′, ‘CIM-616′ and ‘CIM-707′) with variable staple length medium, long and short respectively. The present work aimed to investigate the expression levels of three fiber genes, namely PEPc, XTH, and GA-20 Oxidase, throughout several developmental phases. Results: All three genes have same expression at 15 DPA fibers in all three genotypes but PEPc transcripts are high at 15 DPA stage in 'CIM-707' (long staple length genotype). These fiber development genes may be transformed to the plants with long fiber length through breeding programs. The molecular analysis of fiber development has significant importance in understanding the process of plant cell fate determination, which in turn may contribute to the improvement of fiber production in the long run. The primary impetus behind investigating the molecular mechanisms behind fiber creation is in the pursuit of enhancing cotton fiber quality and yield via prospective endeavors in genetic engineering and breeding.