Background: Treatment of patients with locally advanced rectal cancer (LARC) is based on a combination of chemo-radiotherapy (CRT) and surgery. The rate of distant recurrences remains over 25%. Circulating cell-free DNA (cfDNA) in plasma is a mixture of normal and cancer-specific DNA segments and is a promising biomarker in patients with colorectal cancer. The aim of our study was to investigate plasma cfDNA as a prognostic marker for outcome in patients with LARC treated with neoadjuvant CRT and surgery. Patients and methods: In total, 123 patients with LARC were included in 2 biomarker studies. Patients were treated with neoadjuvant CRT before TME surgery. Fifty-two (42%) of the patients received induction chemotherapy with capecitabine + oxaliplatin. Total cfDNA was measured by direct fluorescent assay in EDTA plasma samples obtained at baseline, after induction chemotherapy, and after CRT. Serial samples 5 years after surgery were collected in 51 patients (41%). Results: Median follow-up was 55months. Distant or local recurrence was seen in 30.9% of the patients. Patients with baseline cfDNA levels above the 75th quartile had a higher risk of local or distant recurrence and shorter time to recurrence compared with patients with plasma cfDNA below the 75th percentile (HR=2.48, 95% CI: 1.3-4.8, P=0.007). The same applied to disease-free survival (DFS) (HR=2.43, 95% CI: 1.27-4.7, P=0.015). Inmultivariate analysis, a high cfDNA level was significantly associated with time to progression and DFS. During follow-up, the association remained significant regardless of time point for sample analysis. Conclusion: We have demonstrated an association between a high baseline plasma level of cfDNA and increased risk of recurrence, shorter time to recurrence, and shorter DFS in patients with LARC. Consequently, cfDNA could potentially improve pre- and post-treatment risk assessment and facilitate individualized therapy for patients with LARC.