Histone variants perform unique functions and are deposited onto DNA by mechanisms distinct from those of canonical histones. The H2A variant, H2A.Z, also known as Htz1 in Saccharomyces cerevisiae, is not uniformly distributed across the genome but facilitates transcriptional activation at target gene promoters and anti-silencing at heterochromatin loci. Htz1 is also involved in DNA replication, DNA repair, chromosome segregation and cell cycle control. Its sequence identity to canonical H2A is only ∼60%, and it is likely that the nonconserved residues are responsible for Htz1-specific functions. However, precise roles of these variant-specific residues are not well understood. To gain insights into the molecular basis underlying the functional differences between canonical and variant histones, 117 alanine-scanning point mutants of Htz1 were constructed for this study, and chemical genetic screens were carried out. Consequently, seven Htz1 residues that conferred one or more abnormal phenotypes when mutated were identified. Based on primary sequence and functional conservation between H2A and Htz1, two of these residues (F32 and I109) appear to have an Htz1-specific role, whereas the rest seem to have functions shared between H2A and Htz1. This study provides a useful resource for future investigations into functional convergence and divergence between canonical and variant histones.