Objective: To determine the value of duplex ultrasound in the detection of significant (⩾50%) stenosis and the location of the stenosis in arteriovenous fistula, compared to angiography. Methods: Patients who underwent construction of an autologous arteriovenous fistula between January 2007 and December 2013 in Treant Care Group, hospital location Emmen, were included in this study. In all patients with a significantly decreased blood flow (flow <400 mL/min and/or ⩾20% decrease) measured by Transonic flowmeter before December 2016, duplex ultrasound was performed. Concordance between duplex ultrasound and angiography was analysed in all patients with a haemodynamically significant stenosis detected by duplex ultrasound. Results: In all, 63 patients had a significant decrease in blood flow leading to duplex ultrasound. In 51 (80.9%) of the 63 duplex ultrasound, a haemodynamically significant stenosis was detected. In 45 (88.2%) of these, angiography was performed, all confirming the presence of significant stenosis. In eight patients, no angiography was performed (sufficient residual blood flow (n = 7), death (n = 1)). Most stenoses were located in the venous outflow tract (75.6%). In 95.6%, a venous approach was possible during angiography. After intervention, a significant increase in blood flow was observed (from 530 mL/min to 910 mL/min (p < 0.001)). Conclusion: We show that duplex ultrasound is likely reliable to ascertain the presence of arteriovenous fistula stenosis in addition to flow criteria. Also, it provides important information to select the most effective and safe approach for cannulation. Duplex ultrasound may reduce costs and burden of diagnosing stenoses.