To identify reliable reference genes for gene expression analysis in Gossypium raimondii. Five different software tools, geNorm, NormFinder, BestKeeper, ReFinder and a dagger Ct method were employed to analyze the qRT-PCR data systematically of 12 housekeeping genes. SAD and TUA11 showed relatively stable expression levels in all tissues (i.e. leaves, shoots, buds, and sepals). We then limited our analysis to each plant part and identified tissue-specific reference genes. Our results showed TUA11, TUB6 and EF1a, EF1a, MZA and GAPC2, MZA, GAPC2, SAD and TUA11, and UBQ and MZA were reliable reference genes in leaves, shoots, buds, and sepals, respectively. Some genes were commonly identified as candidate reference genes in more than two tissue, while others were tissue-specific. Thus, our study allows choosing an appropriate control gene based on sampling for gene expression analysis.