Molecular markers are useful for precise assessment of diversity and phylogenetic relationships among different species and related genera at the DNA level without some of the over simplifying assumptions associated with calculating genetic diversity based on pedigree history. The objective of present study was to have a basic understanding of the genetic diversity that exists in the germplasm available for breeding, which is fundamental for the success of any breeding program. For this study 40 sugarcane accessions were collected from the germplasm source. The accessions consist of advance lines, approved varieties and wild species (S. barberi). Random Amplified Polymorphic DNA (RAPD) and Micro-satellite (SSR) analysis was performed using 80 primers. Use of SSR and RAPD primers together offers the potential of more cost effective data acquisition than is the case with other technologies. These primers sequence vary in different varieties of sugarcane, and this variability may be used to develop molecular markers for mapping sugarcane genes and traits, these sequences being the part of sugarcane genome predicted to be most immediately useful to plant breeder and geneticists.