<h3>Purpose</h3> Cardiac allograft rejection is monitored by histological evaluation of surveillance endomyocardial biopsies (EMB). EMB is invasive and may result in complications, such as bleeding, pneumothorax, damage to tricuspid valve, or cardiac tamponade. Furthermore, the biopsies evaluated by pathologists represent only a very small local histological area and their analysis is imprecise due to inter-observer variance. Donor-derived cell-free DNA (dd-cfDNA) detection from peripheral blood has been previously proposed as a feasible alternative and less-invasive method. The purpose of this study is to validate the method of detecting dd-cfDNA in a prospective patient cohort. <h3>Methods</h3> We have prospectively collected clinical data, EMB, and blood samples from all heart transplant recipients operated in our unit. Patients were treated with tacrolimus-based triple-drug immunosuppression including anti-thymocyteglobulin induction therapy. EMB samples were taken at 2, 4, 6, 8, 10, and 12 weeks, and 4, 5, 6, 8, 10, and 12 months after transplantation and subjected to histological evaluation. Simultaneously, selected single-nucleotide polymorphisms were determined from pre-biopsy peripheral blood samples, and the ratio of dd-cfDNA-to-recipient-cfDNA was calculated. With every EMB sampling, an extra study sample is taken for RNA-sequencing analysis. <h3>Results</h3> We have collected data and samples from 52 consecutive heart transplant recipients with the mean follow-up of 19.2±9.2 months and performed 399 dd-cfDNA quantitations. Of these, 10 results were excluded from the analyses due to multiorgan transplantation (heart+kidney), and 5 results were discarded due to insufficient corresponding EMB sample. Our cohort consists of 297 samples with G0R, 97 with G1R, 8 with G2R, and one with G3R histopathological rejection. The corresponding dd-cfDNA values were 0.16%±0.216% for G0R, 0.16%±0.217% for G1R, and 0.28%±0.162% for G2R/G3R. The predictive value of dd-cfDNA for acute rejection failed to reach statistical significance. <h3>Conclusion</h3> The novel detection method for dd-cfDNA shows promising preliminary results with higher values in more severe histopathological rejection classes. The limitations of our current results stem from the low numbers of G2R/G3R rejections.