Studies have demonstrated that radical esophagectomy can significantly prolong disease-free survival and improve the survival rate of patients with T3 or T4 esophageal cancer and lymph node metastasis. Multidrug resistant cancer cells have active efflux mechanisms that prevent the accumulation of chemotherapeutic drugs in the cells. The purpose of this study was to compare the expression of five MDR related proteins between primary tumors in patients with thoracic esophageal squamous cell carcinoma (ESCC) and metastatic cancer in lymph nodes to explore the clinical significance of heterogeneity in MDR metastatic cancer cells. Fifty-four patients with ESCC and lymph node metastasis were included. All patients underwent subtotal esophagectomy and D2/D3 lymph node resection. The expression of lung resistance-related protein (LRP), P-glycoprotein, topoisomerase-II, thymidylate synthase, and glutathione S-transferase P1-1 (GST-pi) were determined in the primary tumors and lymph nodes via immunohistochemistry. The expression of LRP was significantly different between the primary tumors and lymph nodes (P = 0.026). No significant differences were found for the other four proteins, and protein expression was not associated with either degree of differentiation or disease stage. It was also found that GST-pi was expressed in all patients in both the primary tumors and lymph nodes, suggesting that the design and application of chemotherapeutic protocols capable of reducing GST-pi expression may be beneficial for patients with ESCC. Additional research regarding the clinical utility of MDR protein expression in ESCC is warranted to design effective chemotherapeutic protocols.