Lymphatic dysfunction is involved in many diseases including lymphedema, hypertension, autoimmune responses, graft rejection, atherosclerosis, microbial infections, cancer and cancer metastasis. Expanding our knowledge of lymphatic system function can lead to a better understanding of these disease processes and improve treatment options. Here, optical coherence tomography (OCT) methods were used to reveal intraluminal valve dynamics in 3 dimensions, and measure lymph flow and vessel contraction simultaneously in 3 neighboring lymphangions of the afferent collecting lymphatic vessels to the popliteal lymph node in mice. Flow measurements were based on Doppler OCT techniques in combination with exogenous lymph labeling by Intralipid. Through these imaging methods, it is possible to study lymphatic function and pumping more comprehensively. These capabilities can lead to a better understanding of the regulation and dysregulation of lymphatic vessels in health and disease. The image depicts the dynamic measurements of lymphatic valves, lymphatic vessels cross‐sectional area and lymph velocity simultaneously measured in vivo with optical coherence tomography.