Recurrence after neoadjuvant chemo-radiotherapy (CRT) followed by surgery is high in patients with esophageal cancer. No standard second line therapy is currently available for patients with recurrence. This study aimed to evaluate the expression of chemo-radiosensitive genes after neoadjuvant CRT in residual tumor cells. Thirteen patients with esophageal squamous cell carcinoma underwent 5-fluorouracil (5-FU) and cisplatin (CDDP) based CRT followed by surgery. Total RNA was successfully obtained from 6 formalin-fixed paraffin-embedded (FFPE) specimens using proteinase K digestion and phenol chloroform extraction. TS and DPD as the 5-FU pathway gene, ERCC1 as the CDDP pathway gene, and EGFR, VEGF, HIF1a as radioresistant genes were measured using real-time reverse transcription polymerase chain reaction; comparing the mRNA level of each gene in pre-CRT biopsy with that in post-CRT FFPE specimens. Five patients had less than one-third residual tumor cells in resected specimens histopathologically; eight had more than two-thirds residual tumor cells. There were significant increases in TS (p=0.02) and DPD (p=0.01) levels in residual tumor cells after CRT. Significant decreases in ERCC1 (p=0.03), EGFR (p=0.01), VEGF (p=0.003) and HIF1a (p=0.003) levels were observed. 5-FU and CDDP based CRT up-regulated 5-FU pathway genes and down-regulated CDDP pathway and radioresistant genes. The expression of chemo-radiosensitive genes was significantly changed in residual tumor cells after CRT. Gene expression analysis of residual tumor cells in FFPE specimens may be useful when selecting a second line chemotherapy regimen for recurrent esophageal cancer after CRT.