Objective The aim of this study was to compare the diagnostic accuracy of myocardial perfusion imaging (MPI) by positron emission tomography (PET) with the diagnostic accuracy of MPI by single photon emission computed tomography (SPECT) in two comparable patient cohorts, using coronary angiography (CA) as the standard of reference. Methods A “SPECT-group” of 80 patients (15 female, 65 male; mean age 60 ± 9 years) and a “PET-group” of 70 patients (14 female, 56 male; mean age 57 ± 10 years) underwent a one day stress/rest examination either with attenuation-corrected 13N-ammonia PET or attenuation-corrected 201TlCl SPECT or 99mTc-hexakis-methoxy-isobutyl-isonitril (MIBI) SPECT. PET and SPECT results were semiquantitatively graded using a 6-segment heart model. All patients underwent CA, and stenoses were graded as a diameter reduction ≥50%. Results Coronary findings between both groups did not significantly differ at CA. For the SPECT-group overall sensitivity and specificity for localisation of stenoses was 77% and 84%. Respective values for the PET-group were 97% and 84%. The specificity of MPI by SPECT in the detection of ischemia was 74% and 91% for MPI by PET. The diagnostic accuracy of MPI improves when the individual coronary dominance and previous coronary revascularisations are taken into account. Conclusion MPI by 13N-ammonia PET is more sensitive in the detection and localisation of coronary stenoses, and more specific in the detection of ischemia than MPI by 201TlCl/99mMIBI SPECT.