The purpose of this study was to establish and characterize patient-derived esophageal squamous cell carcinoma xenograft (PDECX) mice for utilization in antitumor drug discovery. A total of 96 esophageal squamous cell carcinoma (ESCC) tissues from Chinese patients were transplanted subcutaneously into immunodeficient mice. Histology, EGFR, K-ras, B-raf, and PIK3CA mutations, and HER2 gene amplifications were analyzed in both patient tumors and mouse xenograft tissues using immunohistochemistry, mutant-enriched liquid chip sequencing and fluorescence in situ hybridization assays, respectively. Furthermore, in vivo efficacy studies using five PDECX mice harboring a variety of genetic aberrations were performed using the chemotherapy agents 5-fluorouracil (5-FU) and cisplatin. Thirty-seven PDECX mouse models were successfully established in immunodeficient mice. Pathological analysis revealed similar histological architecture and degrees of differentiation between patient ESCC and xenografted tumors. No mutations were identified in EGFR, K-ras, and B-raf genes in either xenograft models or patient ESCC tissues. In contrast, PIK3CA gene mutations were detected in 12.5% (12/96) ESCC patients and 18.9% (7/37) PDECX models. Interestingly, patient ESCC tissues exhibiting HER2 overexpression or gene amplification were unable to survive in immunodeficient mice. Further analysis showed that PDECX models carrying HER2 2+ expression had no response to 5-FU/cisplatin, compared with HER2-negative models. In conclusion, a panel of PDECX mouse models, which include PIK3CA mutant and HER2-positive models, was established and characterized thus mimicking the current clinical genetic setting of esophageal carcinoma. The sensitivity of HER2-negative ESCC models to chemotherapy supports stratification approaches in the treatment of esophageal carcinoma patients and warrants further investigation of the impact of PI3KCA on treatment response.