Accurate staging of penile cancer requires invasive methods such as sentinel node biopsy or lymphadenectomy (LAD). We assessed the value of [18F]FDG PET/CT for non-invasive nodal staging in penile cancer (PC) patients before inguinal LAD. 41 consecutive patients with PC (stage pT1 or higher, cN0) received [18F]FDG PET/CT before undergoing bilateral modified or radical inguinal staging LAD. Lymph nodes with a visually increased [18F]FDG uptake were classified as suspicious of lymph node metastases (LNM). Standardized uptake value (SUV) of suspicious inguinal lymph nodes was determined. Results of [18F]FDG PET/CT were correlated with histopathology. In total 623 lymph nodes were resected, in 10 patients LNM were histologically confirmed (14/623 lymph nodes). In patient-based analysis [18F]FDG PET/CT showed a sensitivity and specificity of 80% and 68 %, respectively, a positive predictive value (PPV) of 44 % and a negative predictive value (NPV) of 91 %. In the groin-based analysis, [18F]FDG PET/CT had a sensitivity of 69 %, a specificity of 77 %, a PPV of 36 % and a NPV of 93 %. There was no significant difference in SUVmean and SUVmax between true positive and false positive lymph nodes (p = 0.093 and 0.069, respectively). [18F]FDG PET/ CT shows a high NPV in penile cancer patients without clinically evident LNM. However, due to its limited sensitivity (especially with respect to LNM of small size) and specificity (i. e. in the differentiation between (post)inflammatory and metastatic lymph nodes) [18F]FDG PET/CT cannot replace invasive nodal staging.