Abstract: A RAPD methodology for differentiation and characterization of the fungal edible species Pleurotus ostreatus and Pleurotus sajor-caju was developed. Two of the most usual methods of DNA extraction were studied. The first was based in the simultaneous precipitation of proteins and polysaccharides by the presence of SDS (sodium dodecyl sulphate) and sodium acetate and the second by the use of cetyltrimethylammonium bromide (CTAB). Both methods presented satisfactory results and the best extraction products were obtained by the one that used SDS and sodium acetate. The study with RAPDs produced consistent DNA fragments, reproducible in different gels, serving as good markers for characterization and genetic differentiation of the studied species of mushrooms. The PCR conditions for obtaining RAPD markers were optimized with good interpretation.