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Microarrays have become an important technology for the global analysis of gene expression in organisms. For discovery of differentially expressed genes, many cDNA microarray experiments gave a fixed threshold based on a fold change of signal intensity in relative expression level and/or a P value, the deviation of the fixed threshold could be observed by microarray experiments of self-versus-self hybridization. In order to accurately identify differentially expressed genes in two-channel microarray, control experiments through the self-versus-self hybridizations were performed with wheat cDNA microarrays in this study, distribution of extremes of expression levels was obtained from data analysis of the control experiments, and a threshold level was assigned based on the moment distribution method, the differentially expressed genes can be identified when the changes of expression levels of a gene at different treatments falls outside the threshold. This method could be appropriate to estimate the threshold limits and can be applied for cDNA microarray.