CD133-positive cells have been reported to possess a cancer-initiating-cell phenotype and the property of resistance to chemoradiation therapy in colorectal cancer. The aim of the present study was to evaluate quantitative and locational changes in CD133-positive cells in rectal cancer patients who received preoperative chemoradiation therapy. The prognostic significance of CD133 expression in patients with preoperative chemoradiation therapy was also analyzed. Immunohistochemical staining for CD133 and cancer-initiating-cell marker CD44 were performed in 92 surgically resected rectal cancers. Of the 92 cases, 43 patients received preoperative chemoradiation therapy and 49 patients underwent surgery alone. Forty pretherapic biopsy specimens from 43 patients in preoperative chemoradiation therapy group were also analyzed. CD133-positive cases were more common in the preoperative chemoradiation therapy group than in the surgery-alone group (P = 0.03). Further, CD133-positive cases were more common in the preoperative chemoradiation therapy group than in pretherapic biopsy specimens (P = 0.02). In the preoperative chemoradiation therapy group, the CD133-positive cases showed poorer prognosis than the CD133-negative cases. On the other hand, the frequency of CD44-positive case within cancer tissue was similar between the preoperative chemoradiation therapy group and the surgery-alone group. CD44 expression in the preoperative chemoradiation therapy group was not associated with prognosis. CD44- and CD133-positive cells were distributed evenly within the tumor both in the preoperative chemoradiation therapy group and surgery-alone group, and locational alteration was not observed. The therapy-resistant ability of CD133-positive cells can be associated with poor outcome in the patients with preoperative chemoradiation therapy.