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Abstract Objectives Whether a stenosis can cause hemodynamic lesion-specific ischemia is critical for the treatment decision in patients with coronary artery disease (CAD). Based on coronary computed tomography angiography (CCTA), CT fractional flow reserve (FFRCT) can be used to assess lesion-specific ischemia. The selection of an appropriate site along the coronary artery tree is vital for measuring FFRCT. However the optimal site to measure FFRCT for a target stenosis remains to be adequately determined. The purpose of this study was to determine the optimal site to measure FFRCT for a target lesion in detecting lesion-specific ischemia in CAD patients by evaluating the performance of FFRCT measured at different sites distal to the target lesion in detecting lesion-specific ischemia with FFR measured with invasive coronary angiography (ICA) as reference standard. Methods In this single-center retrospective cohort study, a total of 401 patients suspected of having CAD underwent invasive ICA and FFR between March 2017 and December 2021 were identified. 52 patients having both CCTA and invasive FFR within 90 days were enrolled. Patients with vessels 30%-90% diameter stenosis as determined by ICA were referred to invasive FFR evaluation, which was performed 2–3 cm distal to the stenosis under the condition of hyperemia. For each vessel with 30%–90% diameter stenosis, if only one stenosis was present, this stenosis was selected as the target lesion; if serial stenoses were present, the stenosis most distal to the vessel end was chosen as the target lesion. FFRCT was measured at four sites: 1 cm, 2 cm, and 3 cm distal to the lower border of the target lesion (FFRCT-1 cm, FFRCT-2 cm, FFRCT-3 cm), and the lowest FFRCT at the distal vessel tip (FFRCT-lowest). The normality of quantitative data was assessed using the Shapiro–Wilk test. Pearson's correlation analysis and Bland–Altman plots were used for assessing the correlation and difference between invasive FFR and FFRCT. Correlation coefficients derived from Chi-suqare test were used to assess the correlation between invasive FFR and the cominbaiton of FFRCT measred at four sites. The performances of significant obstruction stenosis (diameter stenosis ≥ 50%) at CCTA and FFRCT measured at the four sites and their combinations in diagnosing lesion-specific ischemia were evaluated by receiver-operating characteristic (ROC) curves using invasive FFR as the reference standard. The areas under ROC curves (AUCs) of CCTA and FFRCT were compared by the DeLong test. Results A total of 72 coronary arteries in 52 patients were included for analysis. Twenty-five vessels (34.7%) had lesion-specific ischemia detected by invasive FFR and 47 vesseles (65.3%) had no lesion-spefifice ischemia. Good correlation was found between invasive FFR and FFRCT-2 cm and FFRCT-3 cm (r = 0.80, 95% CI, 0.70 to 0.87, p 0.05). The AUCs were similar between FFRCT-1 cm + FFRCT-2 cm, FFRCT-3 cm + FFRCT-lowest and FFRCT-2 cm alone (AUC = 0.857, 0.857, 0.857, respectively; p > 0.05 for all). The AUCs of FFRCT-2 cm + FFRCT-3 cm, FFRCT-1 cm + FFRCT-2 cm + FFRCT-3 cm, FFRCT-and 2 cm + FFRCT-3 cm + FFRCT-lowest (0.871, 0.871, 0.872, respectively) were slightly higher than that of FFRCT-2 cm alone (0.857), but without significnacne differences (p > 0.05 for all). Conclusions FFRCT measured at 2 cm distal to the lower border of the target lesion is the optimal measurement site for identifying lesion-specific ischemia in patients with CAD.