The lymphatic network is complex and difficult to visualize in real-time in vivo. Moreover, the direction of flow within lymphatic networks is often unpredictable especially in areas with well-developed "watershed" or overlapping lymphatics. Herein, we report a method of in vivo real-time multicolor lymphatic imaging using cadmium-selenium quantum dots (Qdots) with a fluorescence imaging system that enables the simultaneous visualization of up to five distinct lymphatic basins in real-time. Five visually well-distinguishable carboxyl-Qdots (Qdot 545, 565, 585, 605, and 655) were selected and injected subdermally into mice at five different sites, and serially imaged in vivo or in situ under surgery with real-time multicolor lymphatic imaging. In all seven mice, in vivo lymphatic images successfully distinguished all five lymphatic basins with different colors in real-time. These visualizations of lymph node lasted up to at least 7 days. This method could have a considerable potential in lymphatic research for studying the anatomy and flow within the lymphatic system as well as in some limited clinical settings where real-time visible fluorescence could facilitate procedures under surgery or endoscopy.