Histones bind directly to DNA and play a role in regulating gene expression in part by influencing chromatin structure. The DNA sequences of these histone genes are quite similar, which has hindered individual analyses. The exact function of the 13 different isoforms of histone H2B remains unclear. In this study, we performed a comprehensive gene expression analysis of the H2B isoforms, focusing on tissue specificity. Our results revealed that the H2bc27 gene exhibited brain-specific expression in mice at E14.5. We generated mice lacking the H2bc27 gene using the CRISPR/Cas9 system. While the phenotype of H2bc27 knockout mouse brains was not different from that of wild-type mouse brains, transcriptome analysis indicated that H2bc27 is associated with regulating the expression of several functional genes involved in mouse brain development. The methods used in this study may serve to facilitate comprehensive H2B isoform analysis.