Background Duplex ultrasound (DUS) is reliably used to detect lesions in the peripheral and carotid arterial beds and venous system. Although commonly used in clinical practice, duplex criteria to define lesions in arteriovenous access are not well characterized. This study will define the optimal Doppler-derived peak systolic velocity (PSV) and velocity ratio (VR) to identify >50% lesions in arteriovenous fistulas (AVF) and arteriovenous grafts (AVG). Methods This retrospective analysis includes patients with both DUS and fistulogram within 30 days. DUS-derived PSV and VR were recorded for 3 segments of each access and compared with fistulograms of the same 3 segments of each AV access. Receiver operating characteristic (ROC) was used to determine the optimal DUS criteria for diagnosis of >50% stenosis. Results Fifty pairs of imaging in 40 patients were available for analysis. Mean PSV and VR for segments with greater than 50% stenosis were significantly greater than those without; mean PSV of 480 cm/sec vs. 297 cm/sec (P < 0.001) and mean VR of 3.81 vs. 2.09 (P < 0.001). The ROC analysis demonstrated an optimal PSV of 404 and VR of 2.2 to diagnose >50% stenosis with area under the curve of 0.825 and 0.821 for PSV and VR, respectively. PSV of 500 had sensitivity (Se) of 0.60, specificity (Sp) of 0.86, positive predictive value (PPV) of 0.72, and negative predictive value (NPV) of 0.78. VR of 3.0 had Se of 0.52, Sp of 0.91, PPV of 0.77, and NPV of 0.75. Conclusions DUS-derived PSV of 400 cm/sec and VR of 2.25 have good discrimination to predict greater than 50% stenosis in AVFs and AVGs. Given the broad range of velocities in AV accesses, a threshold of PSV greater than 500 cm/sec and VR greater than 3.0, will reliably identify graft-threatening lesions. Se and Sp of PSV 500 are 0.596 and 0.854, respectively. Se and Sp for VR 3.0 are 0.519 and 0.894, respectively.