AbstractCell-free DNA (cfDNA) has been investigated in acute graft-versus-host disease (aGvHD) following allogeneic cell transplantation (HSCT). Identifying the tissue of origin of cfDNA in patients with aGvHD is relevant particularly when a biopsy is not feasible. We investigate the cfDNA tissue of origin in patients with aGvHD using methylated gene biomarkers. Patients with liver, colon, or skin aGvHD (n = 28) were analyzed. Liver- and colon-derived cfDNA was measured using a colon- (SESN3) and liver (PTK2B)-specific methylation marker with digital droplet PCR. A statistically significant difference (p < 0.001) in PTK2B and SESN3 concentration was observed between patients with colon or liver GvHD and the control group. For SESN3 and PTK2B the area under the curve in the receiver-operating characteristic (ROC) space was 0.952 (95% CI, 0.888–1 p < 0.001) and 0.971 (95% CI, 0.964–1 p < 0.001), respectively. Thresholds to differentiate aGvHD from non-aGvHD in colon were 0 (sensitivity: 0.905; specificity: 0.989) and liver 1.5 (sensitivity: 0.928; specificity: 0.910). Clinical improvement of liver or colon aGvHD resulted in PTK2B and SESN3 reduced concentration. Whereas, in those patients without improvement the PTK2B and SESN3 level remained stable or increased. The PTK2B liver-specific marker and the SESN3 colon-specific marker and their longitudinal analysis might improve aGvHD detection.