The organization of nucleosomes into chromatin and their accessibility are shaped by local DNA mechanics and modulated by histone variants. Conversely, nucleosome positions shape genetic variations, which may originate from mismatches during replication and chemical modification of DNA. To investigate how DNA mismatches and histone variants affect the mechanical stability and the exposure of nucleosomal DNA, we used an optical trap combined with single-molecule FRET, and a single-molecule FRET cyclization assay. We found that a single base-pair mismatch enhances DNA bendability and nucleosome mechanical stability. The increase in force required for DNA unwrapping from the histone core is observed for single base-pair mismatches placed at three tested positions: at the inner turn, at the outer turn, or at the junction of the inner and outer turn of the nucleosome. Yeast nucleosomes are mechanically less stable and more symmetrical in the outer turn unwrapping compared to Xenopus nucleosomes. H2A.Z histone variants display no measurable difference in mechanical unwrapping patterns compared to canonical yeast nucleosomes. The results support a model where nucleosomal DNA accessibility is reduced by mismatches, potentially explaining the preferred accumulation of single nucleotide substitutions in the nucleosome core and serving as the source of genetic variation during evolution and cancer progression.