Deconstruction of plant cell wall biomass is a significant bottleneck to the production of affordable biofuels and bioproducts. Anaerobic fungi (Neocallimastigomycota) secrete the largest known diversity of lignocellulolytic carbohydrate active enzymes (CAZymes) in the fungal kingdom (>300 CAZymes), which unaided can degrade up to 60% of the ingested plant material within the animal digestive tract. However, the specific role of individual enzymes in maintaining hydrolytic efficiency remains unknown due to a lack of genetic tools that facilitate testing of gene function in its natural context. Thus, there is a critical need to create methods that manipulate CAZyme expression and rapidly interrogate gene function in anaerobic fungi to identify targets that will advance biofuel and bioproducts production. By leveraging high quality genome isolations, long-read sequencing, and Hi-C (chromosomal conformation capture) sequencing, we have improved genome assembly by an order of magnitude to generate the first genomes for this family of organisms with chromosomal resolution. We are leveraging these high quality genome assemblies to identify genetic elements such as promoters, centromeric sequences, and to characterize genome stability. We have also developed a suite of genetic reporter proteins to enable genome to phenome studies in anaerobic fungi. Current efforts are aimed at identifying an autonomously replicating sequence to enable faithful plasmid replication during cell division. These efforts are enabling the domestication of non-model anaerobic fungi to be leveraged for a variety of biotechnological applications.