Anaerobic fungi (Neocallimastigomycota) are native to the digestive tracts of ruminant and hindgut fermenting animals where they are central to the digestion of crude lignocellulosic materials. Anaerobic fungi 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. By combining PacBio DNA sequencing and HiC chromatin conformation capture sequencing, we have generated the first chromosome resolved reference genomes for 3 isolates of anaerobic fungi representing two genera. We are leveraging this information to identify key genetic parts for construction of a genetic engineering toolbox for anaerobic fungi. Our work provides insight into the mechanisms that control anaerobic fungal plant-degradation abilities and takes a step towards the development of facile tools to enhance CAZyme activity for efficient plant degradation.