Maize chlorotic dwarf virus (MCDV) encodes a 3C-like protease that cleaves the N-terminal polyprotein (R78) as previously demonstrated. Here, we examined amino acid residues required for catalytic activity of the protease, including those in the predicted catalytic triad, amino acid residues H2667, D2704, and C2798, as well as H2817 hypothesized to be important in substrate binding. These and other residues were targeted for mutagenesis and tested for proteolytic cleavage activity on the N-terminal 78 kDa MCDV-S polyprotein substrate to identify mutants that abolished catalytic activity. Mutations that altered the predicted catalytic triad residues and H2817 disrupted MCDV-S protease activity, as did mutagenesis of a conserved tyrosine residue, Y2774. The protease activity and R78 cleavage of orthologs from divergent MCDV isolates MCDV-Tn and MCDV-M1, and other waikavirus species including rice tungro spherical virus (RTSV) and bellflower vein chlorosis virus (BVCV) were also examined.