Abstract Background Pre-operative chemoradiotherapy (CRT) is the standard treatment in clinical stage T3/4 or node positive rectal cancer. However, there are no established biomarkers that can predict the pathological response and clinical outcome to CRT. Methods Immunohistochemical staining was performed in tissue arrays constructed from core tissue specimens taken before treatment and from operative specimens from 112 patients who received 5-FU based pre-operative CRT and surgery. Expression of Ki67, TS, BAX, EpCAM, p53, p21, EGFR, CD44, CD133, CD166, HIF1α and ALDH1 were assessed and correlated with tumor regression grades and disease free survival. Results Of the 112 patients (M/F 74/38, median age: 62), 20 (17.9%) patients achieved pathologic complete remission (pCR). In analyzing the associations between marker expressions and tumor regression grades, high p21 expression at the pretreatment biopsy was significantly associated with non-pCR (p = 0.022) and poor disease free survival (median DFS - low vs high p21: 75.8 vs 58.1 months, p = 0.002). In the multivariate analysis, high p21 expression level at the pre-treatment biopsy was significantly associated with poor DFS (p = 0.001, HR 6.14; 95% CI 2.03, 18.55). High CD166 expression level at the pretreatment biopsy was also associated with poor DFS (p = 0.003; HR 5.61; 95% CI 1.81, 17.35). Conclusion These show high p21 and CD166 expression at the pretreatment biopsy were associated with tumor regression and poor prognosis in patients treated with 5-FU based CRT. Larger, prospective and functional studies are warranted to determine the role of p21 and CD166 as predictive biomarker of response to CRT.