The median methylation level of OPCML, HOXA9, and HOXD9 in serum cfDNA of CCA was 5.73% (0–44.46%), 1.62% (0–40.97%), and 2.57% (0–50%), respectively, whereas that of other biliary disease group was 0% (0–7.59%), 2.24% (0–16.71%), and 0% (0–7.86%), respectively. This evidence concerns the gene HOXD9 and cholangiocarcinoma.