Abstract Background Intracoronary physiological measurements can be subject to the influence of hydrostatic pressure. Saphenous vein grafts (SVGs) diverge in origin and trajectory from native coronary arteries, which may increase the susceptibility of fractional flow reserve (FFR) measurements to be impacted by the hydrostatic pressures. The precise impact of SVG anatomy on physiological pressure indices remains unknown at present. Purpose We sought to study the impact of SVG anatomy on FFR. Methods Included were symptomatic patients with prior coronary artery bypass grafting and at least one non-occluded SVG, who underwent coronary computed tomography angiography (CCTA) preceding invasive coronary angiography. SVG course and height excursion were reconstructed based on the CCTA images. Similarly to native coronary arteries, the impact of hydrostatic pressure on FFR was calculated by adding 0.077 mmHg to the distal coronary pressure for every millimeter height difference in a supine position between the SVG ostium and the position of the pressure wire tip. Results In total, 74 patients (mean age 71 ± 7 years; 85% male) with 87 SVGs were included. A total of 76 SVGs underwent interrogation by FFR. The height difference between SVG ostium and pressure wire tip position was largest for single SVGs to the circumflex artery (Cx; - 55.1 ± 17.0 mm), followed by sequential SVGs to the Cx (- 53.1 ± 17.7 mm) and to the right coronary artery (RCA; - 36.7 ± 21.6 mm). Corrected FFR was significantly lower as compared to uncorrected FFR in single SVGs to the Cx (0.85 ± 0.17 vs. 0.90 ± 0.18), and sequential SVGs to the Cx (0.92 ± 0.14 vs. 0.96 ± 0.15) and the RCA (0.82 ± 0.20 vs. 0.85 ± 0.21) (P<0.001 for all). Conclusions Hydrostatic pressure related to the course anatomy of SVGs notably affects FFR values, resulting in an overestimation of FFR in SVGs to the Cx and sequential SVGs to the RCA. In these SVGs, FFR values near the cutoff value should be interpreted cautiously.