Endoscopic retrograde cholangiopancreatography with brushed cytology is still the standard method for the diagnosis of extrahepatic cholangiocarcinoma in obstructive jaundice; however, the diagnostic yield is limited. To improve the diagnostic sensitivity, DNA methylation analysis is an attractive candidate, since this may constitute a stable marker in brushed specimens. Therefore, this study aims to evaluate the importance of such epigenetic markers in brushed biliary cells from patients with obstructive jaundice for the diagnosis of extrahepatic cholangiocarcinoma. The cells examined were those that were left over from brushed cytology done during routine endoscopic retrograde cholangiopancreatography of patients with extrahepatic cholangiocarcinoma. The methylation states of HOXA1, RASSF1A, P16, and NEUROG1 genes in extrahepatic cholangiocarcinoma were measured by quantitative methylation-specific polymerase chain reaction and compared between brushed biliary cells and normal gall bladder epithelial cells. The results showed that the sensitivity of the methylation index measurements of HOXA1 and NEUROG1 genes from brushed samples was markedly superior to that of standard cytology. In conclusion, measurement of the DNA methylation status of HOXA1 and NEUROG1 genes in leftover brushed biliary cells might serve as a useful supplement in the detection of malignant biliary obstruction by increasing the sensitivity of diagnosis by routine cytology.