Cholangiocarcinoma (CCA) is a highly lethal hepatobiliary malignancy, with prognosis is influenced by anatomical subtypes and etiological factors. This study successfully established three CCA cell lines: KKU-097, KKU-466, and KKU-610, from the primary tumors of patients in liver fluke-endemic areas. These cells represent the perihilar CCA (pCCA) and intrahepatic CCA (iCCA) subtypes. Comprehensive analyses, including histopathology, molecular profiling, biomarkers, cancer phenotype characterization, and drug sensitivity testing with standard chemotherapeutics, were conducted. Whole-exome sequencing was performed to explore genetic alterations. All three cell lines exhibited adherent growth with an epithelial morphology and positive expression of the bile duct epithelial markers CK-7 and CK-19. Cytogenetic analysis revealed highly complex hypertriploid karyotypes with multiple chromosomal aberrations. Among the cell lines, KKU-610 demonstrated higher growth and invasion rates, whereas KKU-466 and KKU-097 cells exhibited less aggressive phenotypes. Drug sensitivity testing demonstrated relative resistance to gemcitabine as a monotherapy and in combination with cisplatin in all three cells. Genomic profiling identified targetable mutations, highlighting these new cell lines as valuable models for investigating the pathogenesis of CCA and evaluating therapeutic strategies.