Objectives The aim of this prospective multi-center study was to investigate the diagnostic value of myocardial blood flow (MBF) quantification using NaI(Tl)-based single-photon emission computed tomography (SPECT) for determining coronary artery disease (CAD) defined by quantitative coronary angiography (QCA). Background Absolute quantitation of MBF and myocardial flow reserve (MFR) using SPECT is clinically feasible; however, whether flow quantification using NaI(Tl) SPECT is superior to commonly performed SPECT myocardial perfusion imaging (MPI) in determining CAD has not been evaluated. Methods Patients with suspected or known CAD underwent pharmacological stress/rest dynamic SPECT imaging and routine SPECT MPI followed by QCA. Obstructive disease was defined as >= 50% reduction in luminal diameter on QCA. Results One hundred fifty-four patients (462 vessels) were included in the analysis. Obstructive CAD was detected in 76/154 patients (49.4%) and 112/462 vessels (24.2%). Optimal cut-off values were 1.86 mL/min/g for stress MBF and 1.95 for MFR, respectively. Stress MBF and MFR were more sensitive than MPI in both individual patients (stress MBF vs MPI: 81.6% vs 51.3%; MFR vs MPI: 72.4% vs 51.3%) and in coronary vascular regions (stress MBF vs MPI: 78.6% vs 31.3%; MFR vs MPI: 75.9% vs 31.3%; all P < .01). In receiver operating characteristic curve analysis, quantification revealed a significantly greater area under the curve than MPI at the patient (stress MBF vs MPI: 0.761 vs 0.641; MFR vs MPI: 0.770 vs 0.641) and the vessel (stress MBF vs MPI: 0.745 vs 0.613; MFR vs MPI: 0.756 vs 0.613; all P < .05) levels. Integrating quantitative SPECT measures with MPI significantly increased the area under the curve and improved the discriminatory and reclassification capacity. Conclusion Flow quantification using NaI(Tl) SPECT provides superior sensitivity and discriminatory capacity to MPI in detecting significant stenosis. Clinical trial registration NCT03637725.