Cholangiocarcinoma is a malignant neoplasm arising from the epithelial cells lining the biliary ducts and its occurrence can be anatomically classified as within the liver (intrahepatic) or outside the liver (extrahepatic). Extrahepatic cholangiocarcinoma, which can be called as biliary tract cancer (BTC), is the most common form of this malignancy, and its etiology is still unclear. In this study, we tried to elucidate the complicated association between receptor tyrosine kinase (RTK) gene polymorphisms and susceptibility of BTC by analyzing frequency distribution of genotypes and alleles of GRB2-associated-binding protein 1 (Gab1), endothelial growth factor receptor (EGFR), and endothelial growth factor (EGF) and identified potential risk of BTC for people carrying specific genotype of Gab1 and EGFR. Two hundred twenty-five and 300 patients with BTC and cholelithiasis (gallstone (GS)), respectively, and 300 controls matched by age, sex, and ethnicity with patients were recruited from Shengjing Hospital of China Medical University from January 2008 to July 2011 with informed consents. Genomic DNA of BTC group was extracted and purified from formalin-fixed, paraffin-embedded tumor tissue sections using QiAamp DNA FFPE Tissue kit. For GS group and controls, DNA was extracted from peripheral blood leukocytes using genomic DNA extraction kit from Aid Lab. Target genes of RTK family were identified from National Center of Biotechnology Information (NCBI) SNP database and Japanese Single Nucleotide Polymorphisms (JSNP) database. Frequency distribution of genotypes and alleles was analyzed using HapMap Project database. All of the statistical analysis was conducted with SPSS 13.0 software. Eight loci were identified for Gab1 (4), EGFR (3), and EGF (1) as the target single-nucleotide polymorphisms (SNPs) for the association of gene polymorphisms and BTC. A/A genotype and A allele of rs3805246 in Gab1 and G/G genotype and G allele of rs2017000 in EGFR were significantly higher in BTC group than in GS group or controls. After controlling for BMI, age, gender, and smoking habit, patients with "A/A + G/A" had 2.154 times odds to have BTC; as for patients with "A/A" only, they still had 1.976 times odds to have BTC. In the rs2017000 of EGFR, patients with "G/G + G/A" had 1.772 times odds to have BTC, and patients with "G/G" only had 1.530 times odds to have BTC. Furthermore, patients with A/A in rs3805246 and G/G in rs2017000 simultaneously had 1.620 times chance to have BTC than people with other genotypes. This study explored the independent potential effect of EGFR signaling transduction pathway and its downstream element Gab1 and the gene-gene interaction on the disease mechanism of BTC in the perspective of genetics and molecular epidemiology.