||Early diagnosis of prostate cancer (PCa) and patients’ stratification into different risk groups are critical for successful management of the disease. DNA methylation of tumor suppressor genes (TSGs) is a potential source of diagnostic and prognostic biomarkers. The present study focused on identification and validation of novel DNA methylation biomarkers of PCa. During the first stage of experimental work, several TSGs were selected for methylation analysis in prostate tumors followed by evaluation of the most potential biomarkers for noninvasive screening in urine. Although the obtained results were promising, the relatively low specificity for PCa encouraged to search for biomarkers with excelling performance. The second stage of the study began with genome-wide DNA methylation profiling, which led to identification of a set of novel putative biomarkers with diagnostic and/ or prognostic value that might even surpass currently available tools. The prostate cancer dataset of The Cancer Genome Atlas project was utilized as an independent validation cohort. Promoter methylation of the newly identified genes was also demonstrated to be detectable in urine of PCa patients, which shows its potential application for noninvasive testing. Although additional validation is required, the obtained results seem to be rather promising. Protein-coding and miRNA host genes with newly identified methylation differences in PCa might be attractive targets for epigenetic therapeutics.