Aim: Metastasis size in melanoma sentinel lymph nodes (SLNs) is an emerging prognostic factor. Two European melanoma treatment trials include SLN metastasis diameters as inclusion criteria. Whilst diameter estimates are sensitive to the number of sections examined, the level of this bias is largely unknown. We performed a prospective multicentre study to compare the European Organisation for Research and Treatment of Cancer (EORTC) recommended protocol with a protocol of complete step-sectioning. Methods: One hundred and thirty-three consecutive SLNs from seven SLN centres were analysed by five central sections 50 μm apart (EORTC Protocol) followed by complete 250 μm step-sectioning. Results: Overall, 29 patients (21.8%) were SLN-positive. The EORTC Protocol missed eight of these metastases (28%), one metastasis measuring less than 0.1 mm in diameter, seven measuring between 0.1 and 1 mm. Complete step-sectioning at 250 μm intervals (Extensive Protocol) missed one metastasis (3%) that measured less than 0.1 mm. Thirteen treatment courses (34%) performed if inclusion was based on the Combined Protocol would not be performed if assessed by the EORTC Protocol. Thus, 10 patients would be without completion lymph node dissection (EORTC MINITUB study), whilst three patients would not be eligible for anti-CTLA4 trial (EORTC protocol 18071). The corresponding number with the Extensive Protocol would be three; one patient for the MINITUB registration study and two patients for the anti-CTLA4 study. Conclusions: Examining SLNs by close central sectioning alone (EORTC Protocol) misses a substantial number of metastases and underestimates the maximum metastasis diameter, leading to important changes in patient eligibility for various treatment protocols.