AbstractEsophageal squamous cell carcinoma (ESCC) ranks fourth among cancer-related deaths in China due to the lack of actionable molecules. We performed whole-exome and T-cell receptor (TCR) repertoire sequencing on multi-regional tumors, normal tissues and blood samples from 39 ESCC patients. The data revealed 12.8% of ERBB4 mutations at patient level and functional study supported its oncogenic role. 18% of patients with early BRCA1/2 variants were associated with high-level contribution of signature 3, which was validated in an independent large cohort (n = 508). Furthermore, knockdown of BRCA1/2 dramatically increased sensitivity to cisplatin in ESCC cells. 5% of patients harbored focal high-level amplification of CD274 that led to massive expression of PD-L1, and might be more sensitive to immune checkpoint blockade. Finally, we found a tight correlation between genomic and TCR repertoire intra-tumor heterogeneity (ITH). Collectively, we reveal high-level ITH in ESCC, identify several potential actionable targets and may provide novel insight into ESCC treatment.