Monitoring the methylation pattern of a single tumor cell might be important in understanding the mechanism of tumor initiation and progression. In this study, we developed a method based on molecular cloning microarray strategy for analyzing methylation patterns of a single DNA fragment from a group of tumor cells. In the method, a microarray of single monoclones of bisulfate-treated PCR products was fabricated by two-primer hyper-branched rolling circle amplification (HRCA) in polyacrylamide gel, in which a library of the bisulfate-treated PCR products with different methylated status from tumor cells were ligated to circle molecules to form HRCA templates, and one of the HRCA primers was modified with acrylamide on its 5'-end. Due to the diffusion retardation of HRCA products in a polyacrylamide matrix, the HRCA products are localized near their respective templates, and formed to a microarray of monoclones. After the nonimmobilized strands were removed, three pairs of probes were used to detect different CpG sites of each clone simultaneously by hybridization. We successfully analyzed the methylation patterns of P16 gene for three cases of stomach tumor tissues. This method could provide a significant tool in detecting the distribution of cells with. different methylation patterns in one tumor tissue.