Abstract Background The prognostic significance of neoantigens and the understanding of neoantigen-associated heterogeneity in intrahepatic cholangiocarcinoma (iCCA) is still unclear. This study aims to investigate the prognostic role of neoantigens and their intratumor heterogeneity (ITH) in postoperative prognoses of patients with resected iCCA. Methods A cohort enrolled 68 iCCA patients underwent surgical resections was performed whole-exome sequencing to analyze the impact of neoantigens on postoperative survival. Immunohistochemistry for CD4 and CD8 was performed and T cells in tumor were quantified by visually counted. RNA sequencing and TCR sequencing were performed to demonstrate tumor immunogenicity and immune recognition associated with neoantigen heterogeneity. Results The neoantigen intratumor heterogeneity (NeoITH) metric was an independent prognostic factor, with NeoITH-low iCCA associated with improved prognosis in the discovery and the validation cohort. Immunogenetic analyses revealed that NeoITH-low iCCA harbored an increased CD8+ T cell infiltration and higher immunogenicity compared to NeoITH-high iCCA. Characterization on the TCR repertoire identified 36 iCCA-related TCR clusters which were enriched in NeoITH-low tumors. Patients identified as the high number of iCCA-related TCR clusters and NeoITH-low iCCAs had the best postoperative prognosis, and vice versa. A retrospective immunotherapy cohort confirmed that patients with NeoITH-low iCCAs had significant therapeutic advantages then patients with NeoITH-high iCCAs. Conclusions This study provides a novel insight into neoantigen heterogeneity for predicting postoperative survival and immunotherapeutic prognosis in iCCA patients.