Cultivar identification in micro-propagated date palm seedlings is laborious so that application of molecular markers to facilitate and acceleration of the procedure seems inevitable. Given the need for control the originality of micro- propagated date palm seedlings, the aim of this study was evaluation of SSR markers usability to cultivar identification in micro-propagated date palm seedlings. Original samples of Green Ghanami, Red Ghanami, Gantar, Deiry, Ostaemran, Barhi, Medjool, Zahedi and Piarum cultivars were used control. Taking into account the rigidity of leaves and subsequently high consumption of liquid nitrogen to powder leaves, an efficient method for powdering of leaves using Tissue Lyser II instrument was optimized. Eight SSR primer pairs were used for polymerase chain reaction. The Results showed that by using these molecular markers and reliable controls, determination of micro- propagated date palm cultivars is feasible. Clustering of cultivars showed that all of them were differentiated using five SSR primer pairs including mPdCIR025, mPdCIR057, mPdCIR070, PDAG1003 and DP175. Also, barcoding of scored band illustrated that c1 allele (230 to 240 bp) for Piarum cultivar and d3 allele (220 to 230 bp) for Medjool cultivar were exclusive. Totally to make the results referable, cultivar identification diagram was drawn up.