Chlorophyllum molybdites is a basidiomycetous fungus that is commonly found growing in cluster on soil with grasses. In this paper, the molecular identification and optimization of cultural conditions for mycelial growth of C. molybdites were investigated. The genomic DNA of wild fruiting body was isolated, polymerase chain reactionamplified using the following internal transcribed spacer (ITS) primer: ITS 1F(F) 5' CTT GGT CAT TTA GAG GAA GTA A 3' and ITS4B R 5' CAG GAG ACT TGT ACA CGG TCC AG 3', blasted in GenBank database, and confirmed the identity using phylogenetic analysis. The optimal nutritional (indigenous media) and physical requirements (temperature, aeration, and illumination) for mycelial biomass production in liquid culture were also established. The results of BLAST and phylogenetic analyses revealed that the genomic DNA isolated showed 99.76% similarity to C. molybdites (KP012712.1) and 72% bootstrap support in the phylogenetic tree. Mycelia of C. molybdites favorably grew in potato dextrose decoction at pH 5.0-5.5, and when incubated at 24-28°C in an unsealed and either lighted or dark conditions.