At early stages of carcinogenesis, the regulatory regions of some tumor suppressor genes become aberrantly methylated at RCGY sites, which are substrates of DNA methyltransferase Dnmt3. Identification of aberrantly methylated sites in tumor DNA is considered to be the first step in the development of epigenetic PCR test systems for early diagnosis of cancer. Recently, we have developed a GLAD-PCR assay, a method for detecting the R(5mC)GY site in the genome position of interest even at significant excess of DNA molecules with a non-methylated RCGY site in this location. The aim of the present work is to use the GLAD-PCR assay to detect the aberrantly methylated R(5mC)GY sites in the regulatory regions of tumor suppressor genes (brinp1, bves, cacna2d3, cdh11, cpeb1, epha7, fgf2, galr1, gata4, hopx, hs3st2, irx1, lrrc3b, pcdh10, rprm, runx3, sfrp2, sox17, tcf21, tfpi2, wnt5a, zfp82, and znf331) in DNA samples obtained from gastric cancer (GC) tissues. The study of the DNA samples derived from 29 tumor and 25 normal gastric tissue samples demonstrated a high diagnostic potential of the selected RCGY sites in the regulatory regions of the irx1, cacna2d3, and epha7 genes; the total indices of sensitivity and specificity for GC detection being 96.6% and 100%, respectively.