The lymphatic system is essential for fluid regulation and for the maintenance of host immunity. However, in vivo lymph flow is difficult to track in real time, because of the lack of an appropriate imaging method. In this study, we combined macro‐zoom fluorescence microscopy with quantum‐dot (Qdot) optical lymphatic imaging to develop an in vivo real‐time optical lymphatic imaging method that allows the tracking of lymph through lymphatic channels and into lymph nodes. After interstitial injection of Qdots in a mouse, rapid visualization of the cervical lymphatics and cervical lymph nodes was achieved. Real‐time monitoring of the injected Qdots revealed that the cortex of the node enhanced first followed by a net‐like pattern in the central portion of the node. Histology revealed that the rim and net‐like enhancing regions corresponded to the subcapsular sinuses and medullary sinuses respectively. Additionally, multiplexed two‐color real‐time lymphatic tracking was performed with two different Qdots. With this real‐time imaging system, we successfully tracked microscopic lymphatic flow in vivo. This method could have a potential impact for lymphatic research in visualizing normal or abnormal functional lymphatic flows. Published 2012. This article is a U.S. Government work and is in the public domain in the USA.