Cholangiocarcinoma (CCA), a neoplasm arising from biliary epithelial cells, is particularly widespread in Southeast Asia, with northeastern Thailand exhibiting the greatest prevalence attributed to Opisthorchis viverrini infection. This malignancy exhibits considerable molecular heterogeneity, leading to therapeutic resistance and recurrence. Comprehending its molecular mechanisms is essential for enhancing diagnostic and treatment approaches. Our research utilized multi-region LC-MS/MS proteomic analysis to investigate intratumor heterogeneity (ITH) in CCA. We examined 52 tumor areas and 13 neighboring tissues from 13 patients, concentrating on protein profiling, pathway analysis, differential protein expression, and the identification of shared and unique protein signatures. The findings indicated considerable inter-patient proteome variability, characterized by markedly distinct protein expressions among individuals, aligning with prior cancer research. Intra-tumor heterogeneity was apparent, with merely 18 proteins common to all tumor areas and patients, underscoring the intricacy of CCA. Significantly, the common proteins were associated with metabolic reprogramming and oxidative stress pathways, indicating possible indicators and therapeutic targets. This work highlights the significant proteome variability in CCA at both intra-tumor and inter-patient levels, underscoring the necessity for customized therapeutic approaches to tackle the disease's complexity and improve treatment outcomes.