Background Determination of coronary flow reserve (CFR) is increasingly being used in cardiac allograft vasculopathy (CAV). We aimed to identify determinants of CFR in heart transplantation (HT). Methods CFR was measured by transthoracic echocardiography in 119 HT recipients (97 men, 22 women; 50 ± 12 years of age at HT and 8 ± 5 years post-HT). CFR was expressed as the ratio of hyperemic (adenosine infusion at a rate of 0.14 mg/kg) to basal diastolic flow velocity. Rejection scores (RS) on endomyocardial biopsy were calculated. Angiographic CAV was analyzed using a qualitative grading system. The coronary tree was divided into 17 traits and a CAV severity/diffusion index (SDI) was calculated for each patient, summing the scores assigned to all lesions. Results Upon multivariate analysis, CFR was related to CAV ( p = 0.001), interventricular septum thickness ( p = 0.01), ischemic heart disease pre-HT ( p = 0.02) and SDI and SDI/segment number ( p < 0.0001 and p = 0.003, respectively). In patients without CAV, CFR was related only to RS for severe grades ( p = 0.01). Conclusions Left ventricular hypertrophy, CAV and its severity/diffusion independently contribute to reduced CFR. In patients without angiographic CAV, CFR was only independently related to RS. Because a high rejection burden is associated with increased risk of CAV, CFR reduction may be an early marker of CAV. Microvascular dysfunction may contribute to the late morbidity and mortality seen in HT.