Background and Purpose Carotid ultrasound had modest accuracy in the North American Symptomatic Carotid Endarterectomy Trial (NASCET) of carotid endarterectomy in predicting severe carotid stenosis when a 250-cm/s peak systolic velocity (PSV) criterion was applied to different laboratories. We compared the performance of two independent laboratories using similar equipment (ATL-HDI Ultramark 9) but different interpretation criteria. Methods Consecutive patients who underwent both color-coded duplex ultrasound and intra-arterial digital subtraction angiography were studied. PSV was determined with angle correction at the site of the tightest arterial narrowing. Carotid stenosis was measured on angiograms using the North American (N) method. Sensitivity, specificity, and positive (PPV) and negative (NPV) predictive values with 95% confidence intervals were calculated for each laboratory. Results In 87 patients, 174 bifurcations were imaged. A 250-cm/s criterion was the best single predictor of a >70% N stenosis at one laboratory (sensitivity 93% [95% confidence interval, 85 to 101], specificity 86% [76 to 96], PPV 75% [62 to 87], and PPV 96% [90 to 102]) but had modest parameters at the other laboratory (50% [34 to 64], 87%, [77 to 97], 60 [44 to 76], and 91 [82 to 100], respectively). However, the diagnostic criteria routinely used in the second laboratory included different velocity values, which when applied decreased specificity by 17% but increased sensitivity by 35% (85% [74 to 96], 70% [56 to 84], 90% [81 to 99], and 77% [64 to 90], respectively). Conclusions Despite the use of similar equipment, ultrasound grading of carotid stenosis is operator dependent and relies on different and individually validated criteria. Greater sensitivity of ultrasound screening is achieved by applying diagnostic criteria specific to each laboratory. Multicenter studies should use laboratory-specific criteria and a local validation process.