Identification of genes with invariant levels of gene expression is a prerequisite for validating transcriptomic changes accompanying development. Ideally expression of these genes should be independent of the morphogenetic process or environmental condition.We report here the validation of internal control gene i.e.TPS (trehalose 6-phosphate-synthase) in cotton (Gossypium spp), using TaqMan system in quantitative Real Time PCR (qRT-PCR). The Gene expression was tested in five different G. hirsutum cultivars including Coker 312, Acala SJ, ZETA 2, Taghva, Neishabour and a diploid wild type; G. barbadense. Identical amplicons were obtained within these cultivars. No amplifications was achieved when DNA samples from barley (Hordeum vulgare), maize (Zea mays), rice (Oryza sativa), Fig (Ficus carica), pistachio (Pistacia vera), yew (Taxus baccata), wheat (Tirticum aestivum), rose (Rosa hybrida) and soybean (Glycine max) were used as template. Therefore, it was confirmed that the primers and probes designed in this study were specific for the identification and quantification of internal control gene. These results reveal the possibility of using the TPS gene as an internal control in cotton. In another word, this gene would be a suitable candidate as a reference gene in examination of gene expression, detection of transgenic cotton and determining e the zygosity as well as the copy number of the transgene.