Sugarcane (Saccharum officinarum L.) is one of the leading crops in the world. Molecular characterization and molecular marker assisted selection in sugarcane breeding might be a tool for improvement of this crop can assist in increasing sugar production. Like many other plant species, sugarcane tissues contain high levels of polysaccharides and polyphenolic compounds, which present a major contamination problem during DNA purification. High-concentrations of polysaccharides, which co-purify with DNA in normal phenol-chloroform extractions and polyphenols covalently bind to DNA making it useless for most research applications. An rapid, efficient and easy DNA purification procedure for sugarcane is an urgent need in genome mapping and marker assisted selection (MAS) programs. In the present investigation an efficient and easy method was developed for isolation of high quality DNA from meristem cylinder in sugarcane. With the method developed high quality DNA isolation was possible without use of liquid nitrogen, tissue homogenizer and using a simple micro-centrifuge. Isolated DNA well performed for PCR amplification and DNA Fingerprinting using RAPD markers.