Abstract The contribution of promoter DNA methylation to the alteration of caner related gene expression has been well studied, however, these genes can also potentially be altered by chromatin accessibility without involvement of DNA methylation and can be epigenetically inherited. In this study, we used an assay developed in our laboratory (AcceSssIble) that can simultaneously interrogate DNA methylation and chromatin accessibility allowing us to investigate the epigenetic changes in uncultured clear cell renal cell carcinoma (ccRCC) tumors and normal tissue to uncover genes that contribute to ccRCC tumorigenesis. AcceSssIble is both cost-effective and easily analyzed using simple and straightforward computational analysis. Our study revealed significant changes to the epigenome of ccRCC, especially identifying epigenetically up- (160) or down-regulated genes (180), which are dependent on accessibility changes with (30%) or without (70%) DNA methylation involvement, and which were validated by a cross-correlation of the identified genes with RNA-seq and DNA methylation data from larger cohorts of ccRCC samples from The Cancer Genome Atlas (TCGA). In addition, our findings also revealed these sets of genes not found to be commonly mutated in ccRCC and undergo epigenetic changes at higher frequencies than common ccRCC mutations. In addition, pathway analysis suggests that genetic and epigenetic alterations are independent events, and one such example includes a set of changes in HIF1α signaling pathway genes that are independent of the Von Hippel-Lindau (VHL) status. This suggests a novel epigenetic basis for HIF1αs role during tumorigenesis that may be a common occurrence in ccRCC. Thus, the AcceSssIble analysis on ccRCC samples revealed novel candidates for epigenetic driver genes which were previously undetectable by widely used methylation studies. Overall, this study provides a novel approach that can help identify new epigenetic therapeutic targets and treatment strategies complementing current approaches based primarily on the genetic makeup of primary tumors. Citation Format: Elinne Becket, Sameer Chopra, Christopher Duymich, Lin J. Justin, Jueng Soo You, Kurinji Pandiyan, Peter W. Nichols, Kimberly D. Siegmund, Peter A. Jones, Gangning Liang. Identification of epigenetic regulated genes through simultaneous analysis of DNA methylation and chromatin structure in uncultured tumors. [abstract]. In: Proceedings of the AACR Special Conference on Chromatin and Epigenetics in Cancer; Sep 24-27, 2015; Atlanta, GA. Philadelphia (PA): AACR; Cancer Res 2016;76(2 Suppl):Abstract nr B03.