Background: Neoadjuvant chemoradiotherapy (nCRT) prior to surgery in rectal cancer has several adverse effects. Predictive biomarkers for response to nCRT are needed to save patients unnecessary toxicities and to take a timely tailored treatment decision. Epigenetic modifications like DNA methylation patterns have been suspected to be potential predictive biomarkers. Aim: To determine the role of TFAP2E and MLH1 genes' methylation status and microsatellite instability (MSI) in predicting response to 5-fluorouracil – based nCRT in rectal cancer. Methods: DNA was extracted from 80 patients with newly diagnosed stage II / III rectal cancer. The methylation status of TFAP2E and MLH1 genes was determined by pyrosequencing and MSI was determined using 5 micro­satellite loci by conventional polymerase chain reaction and capillary electrophoresis. Results:  The cut-off values for TFAP2E & MLH1 genes' methylation level were 40% and 15% by receiver operating characteristic curve analysis. Hypermethylated TFAP2E and MLH1 gene promotors and MSI were predominant among non-responders (p <0.001, <0.001 and =0.022; respectively). Other factors associated with significantly higher pathological response to nCRT were well/moderately differentiated adenocarcinoma, pretreatment carcinoembryonic antigen level ≤5 ng/ml and rectal tumor ≤5 cm from the anal verge. Conclusion: Hypermethylated TFAP2E and MLH1 gene promotors and MSI in rectal cancer tissue were associated with poor response to 5-fluorouracil – based nCRT. They might be of value in predicting the response of rectal cancer to nCRT and in tailoring its treatment.