Aims: Histopathological examination of sentinel lymph nodes in penile cancer has varied during the first 10 years after introduction of the dynamic sentinel node (SLN) procedure in Denmark, and guidelines have been sparse. The aim of this study was to investigate the impact of rigorous step-sectioning of sentinel lymph nodes in penile cancer and improve guidelines. Methods and results: Seventy-two penile squamous cell carcinoma patients undergoing SLN procedure at a single institution in 2000–2010 were included. The archived lymph node tissues already subjected to a standard pathological examination were retrieved and the initially negative lymph nodes were subjected to an extended step-sectioning procedure. The results were compared to clinical patient outcome from a national database and subsequent pathology reports. The original histopathological examination had detected 26 SLN with metastasis, 21 macro metastases and five micro metastases. The additional step-sectioning procedure of this study generated 4606 slides; seven SLN metastases, two macro metastases and five micro metastases, were detected. One of the macro metastases originated from a patient in which a clinical relapse had occurred in the same groin. None of the other metastases detected in this study showed ipsilateral relapse during follow-up. Conclusion: The results underline the value of our current practice of step-sectioning sentinel lymph nodes in penile cancer and the need for histopathological routines and guidelines. The Danish national guidelines on histopathological handling of sentinel lymph nodes have been adapted to detect any potential clinically relevant metastases.