Grass species, which include the major cereal crops maize, wheat, rice, and sugarcane, are an integral part of our global agriculture and source of food and energy for a growing world population. GRASSIUS was established as a knowledgebase for transcription factors (TFs) and coregulators (CoRegs) in maize and several other species in the grass family. TFs are a primary component of the gene regulatory networks (GRNs) and the underlying gene regulatory grids (GRGs) that govern all aspects of plant growth and metabolism. GRASSIUS is the source for all information pertaining to the maize TFome collection, which serves as a powerful resource for the discovery of GRNs in maize and other cereals [1,2]. Here we describe the release of the GRASSIUS 2.0 knowledgebase (www.grassius.org) with updated data, query, and tool features, as well as the ability to expand to accommodate future datasets. The membership and annotation of all TF and CoReg families has been updated and revised to include gene models from v3, v4, and v5, of the maize B73 genome and recent genome versions of rice, sorghum, Brachypodium, and sugarcane. A translation tool enables cross referencing of Gene IDs between versions of the maize genome. Protein-DNA interactions (PDIs) have been added incorporating results derived from various gene- and TF-centered PDI discovery tools and visualized through a new web-based interface. A filtering tools permits the selection and visualization of PDIs within a ± 2 kb distance from the transcriptional start site (TSS) of a gene of interest. A new BLAST (Basic Local Alignment Search Tool) tool facilitates searching of the maize TFome as well as v3, v4, and v5 TF gene model sets. Lastly, we describe the methodology used to implement GRASSIUS 2.0 which can guide others in developing and updating similar plant gene regulatory knowledgebases.