Cytokinins (CKs) regulate a diverse assortment of processes in plants, including cellular division, vasculature differentiation, and meristem maintenance. CK perception and response is regulated through a two-component signal transduction system, which are highly conserved in bacteria, fungi and plants and allow organisms to sense and respond to diverse stimuli. Our analysis of the maize mutant Hairy Sheath Frayed1 (Hsf1) identified the CK receptor Zea mays Histidine-Kinase1 (ZmHK1) as the underlying gene which linked CK signaling to control of leaf growth and patterning for the first time. Three missense mutations in the CK-binding domain of ZmHK1 define all known Hsf1 alleles. Each causes increased ligand binding affinity and hypersignaling, producing altered leaf morphology in Hsf1 mutants. Using a Saccharomyces cerevisiae reporter strain containing the heterologous ZmHK1 gene, we tested the ability of 18 ZmHK1 missense mutations targeted to the CK-binding domain to promote Hsf1-like CK hypersignaling. Yeast harboring these mutant ZmHK1 genes were grown with and without CKs to analyze their ability to bind and signal. Some mutations led to Hsf1-like hypersignaling, while others produced no change in activity. These targeted amino acid changes are providing insight as to which residues are critical for ligand recognition, binding, and signaling.