Sentinel lymph node (SLN) identification with preoperative image guidance may improve pathological staging of prostate cancer by identifying nodes outside the standard template of dissection. Four anesthetized male dogs received an intra-prostatic injection of Ga-68-labeled tilmanocept. Every 20 min the pelvic lymph nodes were imaged using PET/CT fusion imaging. At 90 min post-injection a prostatectomy and extended lymphadenectomy were completed; ex vivo radioactivity was recorded for each node using a handheld gamma detector, and confirmed by calculation of percent-of-injected dose (%ID) via assay of Ga-68 radioactivity. SLNs were defined as containing >10 % of the maximum %ID. Preoperative PET/CT fusion imaging identified a mean of 4.25 lymph nodes per animal (range 3-7); the mean number of SLN per animal was 4.00 (range 2-6).Of the excised SLNs, 29 % were located in the standard external iliac and obturator distribution. The SLN %ID ranged from 0.07 to 2.40 % (mean 0.744 % ± 0.641 %); SLN ex vivo count rate ranged from 88 to 2,175 cpm (mean 896 ± 715 cpm); and the SLN standardize uptake values (SUVs) ranged from 13 to 237 (mean 79 ± 67).There was a high concordance of PET-CT imaging to SLN activity, with sensitivity of 93 %. In this feasibility study, pelvic SLNs attained SUVs within 60 min. PET/CT effectively identified SLNs with good anatomic specificity, and radioactivity by hand-held detection and scintillation counts demonstrated high concordance with preoperative imaging. Gallium-68-labaled tilmanocept was highly specific for sentinel nodes. Image-guided tumor resection and lymphadenectomy may become a promising future application in urologic oncology and warrants further investigation.