Introduction: Endomyocardial biopsy (EMB) drives rejection diagnosis in pediatric heart transplant patients but is subject to false negatives. Cardiac magnetic resonance imaging (CMR) can detect and quantify fibrosis and edema with T1 and T2 mapping. Overlaying CMR data onto X-ray images may be used for EMB procedural guidance. Hypothesis: Overlaying segmented T1 and T2 map images onto X-ray for EMB guidance may improve diagnosis of rejection in pediatric transplant patients. Methods: 30 pediatric heart transplant patients referred for clinically indicated EMB underwent noncontrast CMR with T1 and T2 mapping prior to EMB. If present, regions of T1 and T2 elevation were segmented and exported as an overlay image. Biopsies were obtained without ( unguided ) and then with ( guided ) CMR overlay with T1 and T2 “hotspots”. Each biopsy specimen received a rejection grade, blinded to guided vs. unguided. As a surrogate for clinical rejection, treatment decisions were made without knowledge of CMR results, and logged as no change, oral therapy augmentation, or IV therapy initiation. Treatment category and pathologic grade were compared among biopsies with Fisher’s exact test and logistic regression. Results: 30 patients (median age 13 years (interquartile range (IQR) 8.8, 16.2), BSA 1.36 m2 (IQR 1.09, 1.86), 50% male) underwent 64 encounters for a total of 199 biopsied specimens (58 guided, 141 unguided). Hotspots were identified in 75% (48/64) of encounters, with 69% (33/48) in biopsied regions of the right ventricle allowing for guidance (25 T1 and 8 T2). Patients with T1 or T2 hotspots providing guided EMB were twice as likely to require any augmentation of therapy compared to those with unguided EMB (38% guided vs 17% unguided, p=0.001). Incidence of pathologic biopsy results (grade > 0) was not significantly different between guided and unguided biopsies from the same patients (41% vs 40%, p =0.99). Conclusions: Patients with T1/T2 hotspots on CMR and guided EMB require rejection treatment more frequently than patients with only unguided biopsies, although biopsy grade did not significantly differ within the same patient. Future work will evaluate relationship of CMR T1 and T2 hotspots to treatment, which may establish CMR as a superior screening tool for rejection.