The accurate assessment of nodal involvement in prostate cancer is crucial to planning treatment, yet there is a shortage of noninvasive imaging techniques capable of visualizing nodal lesions directly. This study demonstrates the feasibility of using recombinant human adenoviral vectors to detect nodal metastases in a human prostate cancer model. This was achieved by the prostate-restricted expression of optical and positron emission tomography (PET) imaging reporter genes by the viral vector coupled with the innate lymphotropic properties of adenovirus. We show that peritumoral administration of these vectors results in the direct detection of reporter gene expression in metastatic lesions within sentinel lymph nodes. Notably, this approach parallels the current lymphoscintigraphy method but enables the direct PET visualization of sentinel lymph node metastases, eliminating the need for invasive lymphadenectomy. These findings may lead to more effective diagnostic and therapeutic strategies for individuals with advanced-stage prostate cancer.