Extraction of RNA from Dioscorea is difficult because of rich mucilage and secondary metabolite content. High-quality RNA is required for RT-PCR and transcriptome analysis. Different protocols and common extraction kits were used for RNA extraction in Dioscorea , but the results were not satisfactory. A CTAB based protocol with lithium chloride precipitation yielded good quality RNA. The RNA isolated using this protocol was successfully used for RT-PCR and transcriptome sequencing experiments. Mucilage content varies at different developmental stages of Dioscorea and the present protocol was effective in isolating RNA from such samples. Although the protocol was originally modified for tuber tissues, it can be used also for extraction of RNA from rhizome, root, shoot and leaf.