Background Detailed visualization of the lymphatic vessels would greatly assist in the diagnosis and monitoring of lymphatic diseases and aid in preoperative planning of lymphedema surgery and postoperative evaluation. Purpose To evaluate the usefulness of photoacoustic imaging (PAI) for obtaining three-dimensional images of both lymphatic vessels and surrounding venules. Materials and Methods In this prospective study, the authors recruited healthy participants from March 2018 to January 2019 and imaged lymphatic vessels in the lower limbs. Indocyanine green (5.0 mg/mL) was injected into the subcutaneous tissue of the first and fourth web spaces of the toes and below the lateral malleolus. After confirmation of the lymphatic flow with near-infrared fluorescence (NIRF) imaging as the reference standard, PAI was performed over a field of view of 270 × 180 mm. Subsequently, the number of enhancing lymphatic vessels was counted in both proximal and distal areas of the calf and compared between PAI and NIRF. Results Images of the lower limbs were obtained with PAI and NIRF in 15 participants (three men, 12 women; average age, 42 years ± 12 [standard deviation]). All participants exhibited a linear pattern on NIRF images, which is generally considered a reflection of good lymphatic function. A greater number of lymphatic vessels were observed with PAI than with NIRF in both the distal (mean: 3.6 vessels ± 1.2 vs 2.0 vessels ± 1.1, respectively; P < .05) and proximal (mean: 6.5 vessels ± 2.6 vs 2.6 vessels ± 1.6; P < .05) regions of the calf. Conclusion Compared with near-infrared fluorescence imaging, photoacoustic imaging provided a detailed, three-dimensional representation of the lymphatic vessels and facilitated an increased understanding of their relationship with the surrounding venules. © RSNA, 2020 Online supplemental material is available for this article. See also the editorial by Lillis and Krishnamurthy in this issue.