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The role of coronary computed tomography angiography (CCTA) derived fractional flow reserve (CT-FFR) in the assessment of non-culprit lesions (NCL) in patients with acute coronary syndrome (ACS) is debated. In this prospective clinical study, a total of 68 ACS patients with 89 moderate (30–70% diameter stenosis) NCLs were enrolled to evaluate the diagnostic accuracy of on-site CT-FFR compared to invasive fractional flow reserve (FFRi) and dobutamine stress echocardiography (DSE) as reference standards. CT-FFR and FFRi values ≤0.80, as well as new or worsening wall motion abnormality in ≥2 contiguous segments on the supplying area of an NCL on DSE, were considered positive for ischemia. Sensitivity, specificity, positive, and negative predictive value of CT-FFR relative to FFRi and DSE were 51%, 89%, 75%, and 74% and 37%, 77%, 42%, and 74%, respectively. CT-FFR value (β = 0.334, p < 0.001) and CT-FFR drop from proximal to distal measuring point [(CT-FFR drop), β = −0.289, p = 0.002)] were independent predictors of FFRi value in multivariate linear regression analysis. Based on comparing their receiver operating characteristics area under the curve (AUC) values, CT-FFR value and CT-FFR drop provided better discriminatory power than CCTA-based minimal lumen diameter stenosis to distinguish between an NCL with positive and negative FFRi [0.77 (95% Confidence Intervals, CI: 0.67–0.86) and 0.77 (CI: 0.67–0.86) vs. 0.63 (CI: 0.52–0.73), p = 0.029 and p = 0.043, respectively]. Neither CT-FFR value nor CT-FFR drop was predictive of regional wall motion score index at peak stress (β = −0.440, p = 0.441 and β = 0.403, p = 0.494) or was able to confirm ischemia on the territory of an NCL revealed by DSE (AUC = 0.54, CI: 0.43–0.64 and AUC = 0.55, CI: 0.44–0.65, respectively). In conclusion, on-site CT-FFR is superior to conventional CCTA-based anatomical analysis in the assessment of moderate NCLs; however, its diagnostic capacity is not sufficient to make it a gatekeeper to invasive functional evaluation. Moreover, based on its comparison with DSE, CT-FFR might not yield any information on the microvascular dysfunction in the territory of an NCL.