The anterolateral thigh (ALT) flap has become a workhorse in reconstructive surgery of the head and neck region and the extremities. However, its inconsistent vascular anatomy and frequent intramuscular course of perforators often cause difficulties during the dissection of this versatile flap. Hence, reliable preoperative perforator mapping and identification of vascular anomalies may render the raising of the flap easier and safer. The aim of this study was to evaluate the use of Color Duplex sonography and whether it allows the distinction between septocutaneous and musculocutaneous perforators. For this purpose, the thighs of 13 patients undergoing reconstruction with ALT flaps were examined preoperatively, and results were compared to intraoperative findings. A total of 30 perforators could be detected preoperatively, of which 29 were confirmed during flap dissection. Preoperative Color Duplex sonography correctly predicted the course of all perforators as either running through the vastus lateralis muscle or the intermuscular septum. In our investigations, Color Doppler sonography had a 96.7% positive predictive value and a 96.7% true positive rate in detecting perforators. Color Duplex sonography is a highly reliable tool in the preoperative assessment of ALT flaps. Localization and course of perforators can be determined accurately and vascular anomalies can be identified. © 2012 Wiley Periodicals, Inc. Microsurgery, 2012.