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The aim of this study was to optimize DNA extraction from legume nodules to obtain large amounts of high-quality genomic DNA.Nodules of different legume species were used. Varied concentrations of guanidine thiocyanate (from 6 mol l−1 to 0·05 mmol l−1), a component of DNAzol, were tested. The quality of DNA extract was determined by PCR–RFLP. The best results were obtained with 0·5 mmol l−1 guanidine thiocyanate, which resulted in greater DNA yield than with higher and lower concentrations or with DNAzol.The procedure using 0·5 mmol l−1 guanidine thiocyanate yields the highest DNA amount when compared with previously described protocols and offers a reliable method to isolate DNA from nodules of different origins.Irrespective of nodule origin, DNA yield was increased significantly, by two (e.g., Vigna nodules) to seven (Acacia auricoliformis nodules) times. In addition, the proposed procedure's costs are lower than those using the DNAzol.