Around 80% of mutations in the PTEN gene have been reported to be associated with diseases such as Cowden syndrome, which is an autosomal dominant disorder associated with an increased risk of developing breast, thyroid, and endometrial neoplasms. Recent studies have also demonstrated that KILLIN, which is located proximally to PTEN, shares the same transcription start site, and is assumed to be regulated by the same promoter, but is transcribed in the opposite direction. In this regard, we postulate that there may be a connection between KILLIN/PTEN genes and breast and thyroid cancers. Using real-time quantitative polymerase chain reaction (qPCR), we found that expression of KILLIN, but not PTEN, was significantly decreased in 23 Chinese women with a personal history of breast and thyroid cancer or a personal history of breast cancer and a family history of thyroid cancer, or vice versa, and at least two persons in the family with thyroid cancer or at a young age <40 years, when compared with healthy controls (P<0.0001). No PTEN mutations were found in these 23 patients. We then developed a simple methylation-sensitive restriction enzyme digestion followed by real-time quantitative assay to quantify plasma methylated KILLIN/PTEN DNA in these patients. Plasma levels of methylated KILLIN/PTEN DNA were significantly increased in these patients when compared with healthy controls (P<0.05). This study shows that plasma methylated KILLIN/PTEN DNA was significantly elevated, suggesting hypermethylation of the KILLIN/PTEN promoter in breast and thyroid cancer patients.