Identification of lymphatics by Indocyanine Green (ICG) lymphography in patients with severe lymphedema is limited due to the overlying dermal backflow. Nor can the method detect deep and/or small vessels. Multispectral optoacoustic tomography (MSOT), a real-time three- dimensional (3D) imaging modality which allows exact spatial identification of absorbers in tissue such as blood and injected dyes can overcome these hurdles. However, MSOT with a handheld probe has not been performed yet in lymphedema patients. We conducted a pilot study in 11 patients with primary and secondary lymphedema to test whether lymphatic vessels could be detected with a handheld MSOT device. In eight patients, we could not only identify lymphatics and veins but also visualize their position and contractility. Furthermore, deep lymphatic vessels not traceable by ICG lymphography and lymphatics covered by severe dermal backflow, could be clearly identified by MSOT. In three patients, two of which had advanced stage lymphedema, only veins but no lymphatic vessels could be identified. We found that MSOT can identify and image lymphatics and veins in real-time and beyond the limits of near-infrared technology during a single bedside examination. Given its easy use and high accuracy, the handheld MSOT device is a promising tool in lymphatic surgery.