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Sequencing of ITS region is one of the candidate markers as a DNA barcoding in Plants. Since sequencing technique is expensive and time consuming, we used PCR-RFLP technique and compared the result of these methods (PCR-RFLP and sequencing) to evaluate the efficiency of PCR RFLP technique in recognition of different species of Tilia from Hyrcanain forest. Electrophoresis pattern of ITS regions of nine species of Tilia were studied by eight restriction enzymes. Results indicated that the EcoRI and BsrBI did not have restriction site in the genus Tilia. Mantel test showed that the dendrogram derived from RFLP and cladogram indicated relatively high congruency (r=0.57). PCoA analysis recognized T. dasystyla, T. hyrcana and T. rubra from Hyrcanian's origin, but it could not separate T. begonifloia from the other hyrcanian species. In this respect, derived results were similar to sequencing one. In conclusion, with regard to less expensive and less time consuming PCR-RFLP technique and high similarity between its result with sequencing, we recommend this method as a simple and economical method with relatively high efficiency studding plant phylogeny.