Aims: To compare the sensitivity and specificity of the recently commercially available FLI-1 monoclonal (FLI-1m) antibody with the currently used antibodies [CD99 and FLI-1 polyclonal (FLI-1p)] in the diagnosis of Ewing's sarcoma/primitive neuroectodermal tumour (EWS/PNET) and to determine the diagnostic value of the EWSR1 (22q12) dual-colour, break-apart rearrangement probe fluorescence in situ hybridization (FISH) technique. Materials and methods: Forty-three cases of well-documented EWS/PNET and 15 non-EWS/PNET cases were retrieved from the archival files. Immunohistochemistry (IHC) for FLI-1p, FLI-1m and FISH analysis was performed. Results: The most sensitive and specific test panel for the diagnosis of EWS/PNET is the combination of CD99 and FLI-1p. FISH had a very high specificity (100%) but only a moderate sensitivity (50%). Conclusion: The combination of CD99 and FLI-1p is the method of choice for the diagnosis of EWS/PNET. EWRS1 (22q12) dual-colour, break-apart rearrangement probe FISH should be used as a confirmatory test in addition to CD99 and FLI1-p due to its high specificity.