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Transcriptome analysis reveals novel insights in the plant responses against biotic and abiotic stresses. cDNA-AFLP is appropriate and repeatable method for genome wide expression analysis when genomic information is limited. Although several factors may affect sensitivity, accuracy and repeatability of cDNA-AFLP technique so attention to these factors increases quality of results. In this study cDNA-AFLP technique was optimized for analysis of potato against <em>Ralstonia solanacearum</em> under <em>in vitro </em>conditions through examining various factors and their interactions, that affecting the results of such analysis. Results showed that RNA extraction method and the use of mRNA has large effects on the reliability of results. RNAs extracted based on using a purification column were monotonous. Ribosomal RNA and inhibitors alleviated by mRNA extraction, Also optimization of factors such as concentrations of template RNA, Mgcl₂ and <em>Taq</em> DNA polymerase, in pre and selective amplification has significant effects on number, clarity and concentration of amplificated cDNA fragments.