Lignocellulosic biomass represents an abundant, renewable resource that can be used to produce biofuels, low-cost livestock feed, and high-value chemicals. The potential of this bioresource has led to intensive research efforts to develop cost-effective methods to break down lignocellulose. The efficiency with which the anaerobic fungi (phylum Neocallimastigomycota) degrade plant biomass is well recognized and in recent years has received renewed interest. Transcriptomics has been used to identify enzymes that are expressed by these fungi and are involved in the degradation of a range of lignocellulose feedstocks. The transcriptome is the entire complement of coding and non-coding RNA transcripts that are expressed by a cell under a particular set of conditions. Monitoring changes in gene expression can provide fundamental information about the biology of an organism. Here we outline a general methodology that will enable researchers to conduct comparative transcriptomic studies with the goal of identifying enzymes involved in the degradation of the plant cell wall. The method described will include growth of fungal cultures, isolation and sequencing of RNA, and a basic description of data analysis for bioinformatic identification of differentially expressed transcripts.