Purpose:To evaluate in vivo sentinel lymph node (SLN) mapping by using photoacoustic and ultrasonographic (US) imaging with a modified clinical US imaging system. Materials and Methods: Animal protocols were approved by the Animal Studies Committee. Methylene blue dye accumulation in axillary lymph nodes of seven healthy Sprague-Dawley rats was imaged by using a photoacoustic imaging system adapted from a clinical US imaging system. To investigate clinical translation, the imaging depth was extended up to 2.5 cm by adding chicken or turkey breast on top of the rat skin surface. Three-dimensional photoacoustic images were acquired by mechanically scanning the US transducer and light delivery fiber bundle along the elevational direction. Results: Photoacoustic images of rat SLNs clearly help visualization of methylene blue accumulation, whereas coregistered photoacoustic/US images depict lymph node positions relative to surrounding anatomy. Twenty minutes following methylene blue injection, photoacoustic signals from SLN regions increased nearly 33-fold from baseline signals in preinjection images, and mean contrast between SLNs and background tissue was 76.0 ± 23.7(standard deviation). Methylene blue accumulation in SLNs was confirmed photoacoustically by using the optical absorption spectrum of the dye. Three-dimensional photoacoustic images demonstrate dynamic accumulation of methylene blue in SLNs after traveling through lymph vessels. Conclusion: In vivo photoacoustic and US mapping of SLNs was successfully demonstrated with a modified clinical US scanner. These results raise confidence that photoacoustic and US imaging can be used clinically for accurate, noninvasive imaging of SLNs for axillary lymph node staging in breast cancer patients.