In the present study, we studied the relationship between plasma cell-free DNA and the effect of preoperative chemoradiotherapy in patients with rectal cancer. The concentration, KRAS mutation and O6-methylguanine-DNA methyltransferase (MGMT) promoter methylation status of cell-free DNA were measured by using polymerase chain reaction (PCR) analyses. The response to chemoradiotherapy was assessed using tumor regression grading (TRG) scores. The cell-free DNA concentrations in patients with rectal cancer (n=34) were significantly higher compared to healthy controls (n=10). The 400-base pair (bp) DNA concentration, 400-/100-bp DNA ratio decreased significantly after chemoradiotherapy in the good response group. The incidence of KRAS mutation decreased significantly after chemoradiotherapy in both good and poor response groups. Higher MGMT promoter methylation status at baseline DNA was associated with a better tumor response. Therefore, cell-free DNA detection may be useful in evaluating the effect of preoperative chemoradiotherapy in patients with rectal cancer.