Despite the growing evidence demonstrating that TWIST1 is a noteworthy tumor biomarker, little is known about the clinical significance of TWIST1 methylation in human primary pancreatic cancer. In the present study, the association of TWIST1 methylation with clinicopathological characteristics was examined in human primary pancreatic cancer. Primary pancreatic cancer specimens and corresponding healthy pancreatic non-tumorous tissues from 33 patients with pancreatic cancer were used. Methylation levels of TWIST1 were compared with clinicopathological characteristics. The TWIST1 methylation level was higher in pancreatic cancer compared to corresponding non-neoplastic pancreatic tissues. The mean TWIST1 methylation was 66.7% for pancreatic cancer tissue and 15.0% for corresponding nonneoplastic pancreatic tissue (P=0.0004). These results suggested that TWIST1 methylation is a useful biomarker for the screening of pancreatic cancers. Studies using independent data sets are required to confirm these findings.