Objective: Surgical resection is the mainstay of treatment in biliary tract cancers. However, the prognostic factors after resection are not very well defined. Cytokeratins are a group of intermediate filaments present in epithelial tissues that play a crucial role in cell differentiation. Cytokeratin 20 (CK20) is frequently expressed in different types of cancers, but its effect on survival varies considerably. The current study aimed at assessing the prognostic factors as well as CK20 expression for survival outcomes, which has not yet been studied in this group of patients. Material and Methods: We performed a multicenter, retrospective analysis of patients diagnosed with biliary tract cancer and treated them with upfront surgery. In total, 81 patients from four oncology centers were involved in this study. We aimed to determine the impact of known clinicopathologic factors and CK20 expression on survival rates using Cox regression analysis. Results: Median follow-up was 21.6 months. Surgical margin status and lymph node metastases were independent factors for both disease-free (p=0.006 and p<0.001, respectively) and overall survival (OS) (p=0.004 and p<0.001, respectively), while perineural invasion (PNI) significantly influenced disease-free survival (DFS) (p=0.013). Patients with CK 20 expression had better DFS and OS; however, its prognostic effect was not demonstrated in multivariate analysis (p>0.05). Conclusion: Our study demonstrated that lymph node metastasis, positive surgical margins, and PNI were found to be potential prognostic factors for survival outcomes, whereas the CK20 expression was not precise enough to be considered as an independent factor affecting the prognosis.