Purpose To determine the lymphatic drainage pattern using SPECT/CT in clinically node-negative (cN0) patients with vulvar cancer, and to evaluate the possible implications for the extent of inguinal lymph node dissection. Methods A total of 83 patients with vulvar cancer scheduled for sentinel node (SN) biopsy were injected peritumorally with radioactive nanocolloid particles followed by lymphoscintigraphy and SPECT/CT for anatomical localization. The SN and higher-echelon nodes on SPECT/CT were located in different zones in the groin and pelvic region. The groin was divided into five zones according to Daseler et al.: four zones obtained by drawing two perpendicular lines over the saphenofemoral junction and one zone directly overlying this junction. The nodes in the pelvic region were classified into three zones: external iliac/obturator, the common iliac and the paraaortic zones. Results A total of 217 SNs and 202 higher-echelon nodes were localized on SPECT/CT. All SNs were located in the five zones according to Daseler et al.: 149 (69 %) in the medial superior region, 31 (14 %) in the medial inferior region, 22 (10 %) in the central region, 14 (6.5 %) in the lateral superior region and only 1 (0.5 %) in the lateral inferior region. The higher-echelon nodes were located both in the groin (15 %) and in the pelvic region (85 %). Conclusion In patients with cN0 vulvar cancer, lymphatic drainage occurs predominantly to the medial regions of the groin. Drainage to the lateral inferior region of the groin is only incidental and in SN-positive patients this zone might be spared in subsequent extended lymph node dissection. This may lead to a decrease in the morbidity associated with this procedure. SPECT/CT is able to personalize lymphatic mapping, providing detailed information about the number and anatomical location of SNs for adequate surgical planning in the groin.