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To assess the diagnostic potential of multi-slice single-photon emission computed tomography/computed tomography (SPECT/CT) for preoperative sentinel node (SN) mapping in early stage head and neck squamous cell carcinoma (HNSCC). Retrospective case-control study including data of consecutive HNSCC patients treated between November 2011 and December 2015. The diagnostic accuracy of multi-slice SPECT/CT was assessed with regard to the gold standard intraoperative gamma ray detection probe, using McNemar's test and calculating the area under the ROC curve. Additionally, the hot spot yield of SPECT/CT and planar lymphoscintigraphy (LS) was compared. Compared to the intraoperative gold standard, SPECT/CT showed an overall positive predictive value of 60.3% [confidence interval (CI) 46.6-73.0%)], a negative predictive value of 96.3% (CI 93.6-98.1%), and an accuracy of 90.8% (CI 89.1-92.4%). SPECT/CT detected more hot spots than LS and provided detailed anatomical information as well as relevant additional findings with potential impact on further patient management. Sentinel lymph node biopsy proved to be a reliable and safe procedure with an excellent SN excision rate (97%). Multi-slice SPECT/CT is a highly accurate diagnostic test and matches the gold standard intraoperative gamma ray detection probe.