The cell adhesion molecules CD44v6 and integrin-β 1 are associated with the progression and metastasis of cancer.A novel triplex real-time reverse transcription polymerase chain reaction (qRT-PCR) assay was developed to quantify CD44v6 and integrin-β 1gene expression in peripheral blood mononuclear cells from 30 pancreatic cancer (PC) patients and 12 healthy individuals.The standard curve of the triplex qRTPCR was constructed by optimizing the reaction condition and the amplification efficiency was 102.5, 101.1, and 100.6 % for CD44v6, integrin-β1 and endogenous gene (β-actin) amplification.Nonspecific bands were not observed from the triplex qRT-PCR amplification and the detection limit of this assay was 100 copies.Expression levels of CD44v6 and integrin-β 1 gene were significantly lower in healthy individuals than PC patients (P<0.05).CD44v6 and integrin-β1 gene expression were not associated with the sex, age, and tumor position in PC (P>0.05).CD44v6 gene expression was significantly associated with clinical stage, liver metastasis, and tumor size (P< 0.05).Integrin-β 1 gene expression was significantly associated with clinical stage and liver metastasis (P<0.05).This triplex qRT-PCR assay may provide a useful tool for diagnosis, prognosis, and therapeutic evaluation in PC.