Standard treatment of rectal cancer patients comprises preoperative chemoradiotherapy followed by radical surgery. However, clinicians are faced with the problem that response rates vary from one individual to another. Predictive biomarkers would therefore be helpful. To identify genomic imbalances that might assist in stratifying tumors into responsive or nonresponsive categories, we used metaphase comparative genomic hybridization to prospectively analyze pretherapeutic biopsies from 42 patients with locally advanced rectal cancers. These patients were subsequently treated with 5-fluorouracil-based preoperative chemoradiotherapy. Based on downsizing of the T-category, 21 rectal cancers were later classified as responsive, while the other 21 were nonresponsive. Comparing these two groups, we could show that gains of chromosomal regions 7q32 approximately q36 and 7q11 approximately q31, as well as amplifications of 20q11 approximately q13, were significantly associated with responsiveness to preoperative chemoradiotherapy (P<0.05). However, the probability of detecting these copy number changes by chance is high (P=0.21). Our primary results suggest that pretherapeutic evaluation of chromosomal copy number changes may be of value for response prediction of rectal cancers to preoperative chemoradiotherapy. This will require validation in a larger cohort of patients.