Abstract: Bisulfite restriction analysis (COBRA) combined with bisulfite sequencing (BS) were firstly carried out to screen locus-specific DNA methylation markers in donor cell lines for improving porcine somatic cell cloning efficiency. DNA methylation polymorphisms of eight differentially methylated regions (DRMs) among six donor cell lines and their potential associations with porcine cloning achievements were analyzed. Result indicated that all DMRs presented relatively high degree of DNA methylation polymorphisms, except for XIST -DMR2 and H19 -DMR3 with low extent of DNA methylation variations below 10%. Moreover, three DMRs including LINE1-DMR1, POU5F1-DMR1, and NANOG-DMR1 presented the most DNA hypomethylated status in the most efficient cell line, and thus could be considered as potential DNA methylation genetic markers for selecting optimum donor cell lines for porcine somatic cell cloning.