To evaluate the accuracy of myocardial perfusion SPECT (MPI) in the detection and allocation of vessel specific perfusion defects (PD) using standard distribution territories in a routine clinical procedure of patients with multivessel disease (MVD). Combined quantitative coronary angiography and fractional flow reserve (QCA/FFR) measurements were used as invasive reference standard. 216 vessels in 72 MVD patients (67 ± 10 years, 28 female) were investigated using MPI and QCA. FFR of 93 vessels with intermediate stenoses was determined. MPI detected significant stenoses according to QCA/FFR findings with a sensitivity of 85%. However, vessel-based evaluation using standard myocardial distribution territories delivered a sensitivity of only 62% (28 MPI+ out of 45 (QCA/FFR)+ findings), with specificity, PPV and NPV of 90, 62 and 90%. 7/17 false positive and 7/17 false negative findings (41%) could be attributed to incorrect allocation of reversible PD to their respective coronary arteries. 6/17 (35%) perfusion territories were classified as false negative when additional fixed PD were present. MPI had reasonable sensitivity for the detection of significant coronary artery disease in patients with multivessel disease. However, sensitivity decreased markedly, when the significance of each individual stenosis was evaluated using standard myocardial supplying territories. In this setting, 41% of false negative and false positive MPI findings resulted from incorrect allocation of reversible perfusion defects to their determining supplying vessel.