Purpose: Esophageal squamous cell carcinoma (ESCC) is the most common type of esophageal cancer in China and the 5-year mortality rate is up to 70%. Studies on the ESCC genetic landscape are needed to further explore clinical therapeutic strategies. In this study, we evaluated the genetic landscape of ESCC to aid the search for clinical therapeutic strategies. Patients and Methods: A total of 225 ESCC patients were enrolled in this study. Deep sequencing of 450 cancer genes was performed on formalin-fixed paraffin-embedded tumor biopsies and matched blood samples from patients. Tumor mutational burden (TMB) was calculated using an algorithm developed in-house. Results: Our results showed that the most commonly mutated genes in ESCC were TP53 (96%), CCND1 (46%), FGF4 (44%), FGF19 (44%), FGF3 (44%), CDKN2A (31%), PIK3CA (26%), NOTCH1 (24%), KA4T2D (18%), FAT1 (16%), and LRP1B (16%). We found that TMB correlated with patient drinking status. We identified mutations associated with sex, early ESCC, high TMB, and metastasis lymph nodes. KMT2D mutations associated with sex (P = 0.035), tumor stage (P = 0.016), high TMB (P = 0.0072), and overall survival of patients (P = 0.0026). SPEN mutations associated with high TMB (P = 0.0016) and metastasis-positive lymph nodes (P = 0.027). These results suggested that SPEN and KMT2D could be potential prognosis biomarkers for Chinese patients with ESCC. We also found that the number of positive lymph nodes was associated with disease-free survival. Clinical target gene analysis indicated that nearly half of Chinese ESCC patients might benefit from treatment with gene-specific target drugs. Conclusion: Our study revealed the ESCC mutational landscape in 225 Chinese patients and uncovered the potential prognosis biomarker for Chinese patients with ESCC.