Gene expression in eukaryotic cells is intricately regulated by chromatin structure and various factors, including histone proteins. In Saccharomyces cerevisiae, transcriptionally silenced regions, such as telomeres and homothallic mating (HM) loci, are essential for genome stability and proper cellular function. We firstly observed the defective HM silencing in alanine substitution mutant of 80th threonine residue of histone H3 (H3T80A). To identify which properties in the H3T80 residue are important for the HM silencing, we created several substitution mutants of H3T80 residue by considering the changed states of charge, polarity, and structural similarity. This study reveals that the structural similarity of the 80th position of H3 to the threonine residue, not the polarity and charges, is the most important thing for the transcriptional silencing in the HM loci.