Technical developments in near-infrared fluorescence (NIRF) imaging and tomography have enabled recent translation into investigational human studies. Noninvasive imaging of the lymphatic vasculature for diagnosis and assessment of function has been uniquely accomplished with NIR using indocyanine green (ICG), a nonspecific dye that has comparatively poor fluorescent properties compared to emerging dyes. Adjunct use of NIRF-ICG for (a) intraoperative sentinel lymph node mapping for cancer staging, (b) video-angiography during surgery, and (c) discrimination of malignant from benign breast lesions detected by mammography and ultrasongraphy also evidences the clinical utility of NIRF. Future NIRF imaging agents that consist of bright fluorescent dyes conjugated to disease-targeting moieties promise molecular imaging and image-guided surgery. In this review, emerging NIRF imaging is described within the context of nuclear imaging technologies that remain the “gold standard” of molecular imaging.