The centromere, as an essential element to mediate chromosome segregation, is epigenetically determined by CENH3‐containing nucleosomes as a functional marker; therefore the accurate deposition of CENH3 is crucial to chromosome transmission. We characterized the deposition of CENH3 in maize by over‐expression and mutational analysis. Our results revealed that over‐expressing CENH3 in callus is lethal while over‐expressing GFP‐CENH3 and CENH3‐YFP in callus and plants is not and can be partly deposited normally. Different mutations of GFP‐CENH3 demonstrated that CENH3‐Thr4 in the N terminus was needed for the deposition as a positive phosphorylation site and the last five amino acids in the C terminus are necessary for deposition. The C terminal tail of CENH3 is confirmed to be responsible for the interaction of CENH3 and histone H4, which indicates that CENH3 maintains deposition in centromeres via interacting with H4 to form stable nucleosomes. For GFP‐CENH3 and CENH3‐YFP, the fused tags at the termini probably affect the structure of CENH3 and reduce its interaction with other proteins, which in turn could decrease proper deposition. Taken together, multiple amino acids or motifs were shown to play essential roles in CENH3 deposition, which is suggested to be affected by numerous factors in maize.