STAT3 is an oncogene that regulates critical cellular processes and whose constitutive activation has been demonstrated to correlate with biological and clinical features in many types of human malignancy. In this study, STAT3 activation was assessed in the Ewing sarcoma family of tumours (ESFT), which is characterized by fusion of the EWS gene with one of several Ets transcription factors, most commonly EWS-FLI1. STAT3 activation was assessed by immunohistochemistry using a monoclonal antibody specific for tyrosine(705)-phosphorylated STAT3 (pSTAT3(tyr705)) and a tissue microarray containing 49 paraffin-embedded ESFT tumours with known EWS translocations. Twenty-five (51%) tumours were pSTAT3(tyr705)-positive, as defined by more than 10% tumour cell immunostaining. STAT3 activation correlated with tumour site at presentation, with pSTAT3(tyr705)-negative ESFT involving axial sites predominantly (p = 0.008). Notably, among 31 patients who presented with localized disease, high-level STAT3 activation correlated with better overall survival (p = 0.02). STAT3 activation was not directly related to EWS-FLI1 expression, since EWS-FLI1 transfection did not result in STAT3 activation. Furthermore, detailed molecular analysis indicated that STAT3 activation may be seen with EWS-FLI1 or EWS-ERG and appears to be independent of EWS-FLI1 fusion type. In conclusion, STAT3 activation is present in approximately half of ESFT and correlates with clinical features. The role of STAT3 activation in ESFT pathogenesis seems to be independent of the type of EWS/Ets translocation.