Purpose: To noninvasively map sentinel lymph nodes(SLNs) and lymphatic vessels in rats in vivo by using dual-modality nonionizing imaging - volumetric spectroscopic photoacoustic imaging,which measures optical absorption,and planar fluorescence imaging,which measures fluorescent emission-of indocyanine green (ICG). Materials and Methods: Institutional animal care and use committee approval was obtained. Healthy Sprague-Dawley rats weighing 250-420 g (age range, 60-120 days) were imaged by using volumetric photoacoustic imaging(n = 5) and planar fluorescence imaging (n = 3) before and after injection of 1 mmol/L ICG. Student paired t tests based on a logarithmic scale were performed to evaluate the change in photoacoustic signal enhancement of SLNs and lymphatic vessels before and after ICG injection. The spatial resolutions of both imaging systems were compared at various imaging depths (2-8 mm) by layering additional biologic tissues on top of the rats in vivo. Spectroscopic photoacoustic imaging was applied to identify ICG-dyed SLNs. Results: In all five rats examined with photoacoustic imaging, SLNs were clearly visible, with a mean signal enhancement of 5.9 arbitrary units (AU) ± 1.8 (standard error of the mean) (P <.002) at 0.2 hour after injection, while lymphatic vessels were seen in four of the five rats, with a signal enhancement of 4.3 AU ± 0.6(P =.001). In all three rats examined with fluorescence imaging, SLNs and lymphatic vessels were seen. The average full width at half maximum (FWHM) of the SLNs in the photoacoustic images at three imaging depths(2, 6, and 8 mm) was 2.0 mm ± 0.2 (standard deviation), comparable to the size of a dissected lymph node as measured with a caliper. However, the FWHM of the SLNs in fluorescence images widened from 8 to 22 mm as the imaging depth increased, owing to strong light scattering. SLNs were identified spectroscopically in photoacoustic images. Conclusion: These two modalities, when used together with ICG, have the potential to help map SLNs in axillary staging and to help evaluate tumor metastasis in patients with breast cancer.