Acquired resistance to aromatase inhibitors (AIs) remains a major clinical problem in the treatment of estrogen receptor-positive (ER+) breast cancer. We and others have previously reported widespread changes in DNA methylation using breast cancer cell line models of endocrine resistance. Here, we show that the histone variant HIST1H2BE is hypomethylated in estrogen deprivation-resistant C4-12 and long-term estrogen-deprived (LTED) cells compared with parental MCF-7 cells. As expected, this hypomethylation associates with increased expression of HIST1H2BE in C4-12 and LTED cells. Both overexpression and downregulation of HIST1H2BE caused decreased proliferation in breast cancer cell lines suggesting the need for tightly controlled expression of this histone variant. Gene expression analysis showed varied expression of HIST1H2BE in a large panel of breast cancer cell lines, without restriction to specific molecular subtypes. Analysis of HIST1H2BE messenger RNA (mRNA) expression in ER+ AI-treated breast tumors showed significantly higher expression in resistant (n = 19) compared with sensitive (n = 37) tumors (p = 0.01). Using nanostring analysis, we measured expression of all 61 histone variants in endocrine-resistant and endocrine-sensitive tumors. We found significant overexpression of 22 variant histone genes in tumors resistant to AI therapy. In silico The Cancer Genome Atlas (TCGA) analysis showed frequent amplification of the HIST1 locus. In summary, our studies show, for the first time, that overexpression of histone variants might be important in endocrine response in ER+ breast cancer, and that overexpression is at least in part mediated via epigenetic mechanisms and amplifications. Future studies addressing endocrine response should include a potential role of these currently understudied histone variants.