Purpose: We evaluated the reproducibility of lymphoscintigraphy in the assessment of the location and number of sentinel nodes in patients with penile carcinoma. Materials and Methods: A total of 20 patients were prospectively included in analysis. Lymphoscintigraphy was performed after intradermal injection of 99mtechnetium nanocolloid around the tumor or excision scar. We performed 10-minute anterior dynamic imaging, and static anterior and lateral images were obtained at 30 minutes and 2 hours. The following day scintigraphy was repeated after a second injection of the radiolabeled colloid given in an identical fashion, preceded by acquisition of a starting image. An observer evaluated the paired images and count rates were calculated from the images. Results: At least 1 sentinel node was visualized in all patients on the first lymphoscintigram. A total of 56 sentinel nodes were seen in 38 basins. Drainage to both groins was seen in 18 patients. In 1 of these patients drainage to the prepubic area was also observed. There were 2 patients with drainage to 1 groin. The second lymphoscintigram revealed the same drainage pattern in all patients- the same number of nodal basins and number of sentinel nodes were visualized at identical locations. All hotspots that were visualized during the first lymphoscintigram showed an unequivocal increase in radioactivity after repeat injection. Thus, the reproducibility of penile lymphoscintigraphy was 100% (95% CI 85%-100%). The Pearson correlation coefficient of the paired count rates was 0.69 (p <0.0001). Conclusions: Results of lymphoscintigraphy in patients with penile carcinoma are highly reproducible for assessment of the number and location of sentinel nodes.