This study was accomplished to study genetic variation of 35 cultivars sugarcanes using RAPD marker. The aim was to determine the genetic variation level and relation in some commercial varieties of S.officinarum and the two wild varieties of S.spontneum as well. Total genomic DNA of each cultivar was extracted from 1.5-2.5gr new germinated fresh young leaves and solved in the suitable amount of TE buffer. The concentration was determined by spectrophotometer. RAPD analysis was performed by use of 10bp randomized nucleotide primers. All genotypes were marked for presence or absence of band, and analyzed by use of statistical software NTSYS 2002. Among 65 applicated primers, 30 primers showed polymorphism between cultivars, and 464 polymorphic bands were produced. Cluster analysis lays varieties into the 2 separated groups through the Jacard coefficient and UPGMA methods. In this classification, 2 wild varieties from the S.spontanenm were separated completely and showed quite different band pattern in comparison with other varieties. On the whole, the results of this research showed that RAPD marker is a suitable tool to study genetic variation in sugarcane. It is recommended to increase the efficiency of breeding programs to study precise genetic variation and to determine the relationship among cultivars.