Ewing sarcoma (ES) is a primary bone tumor initiated by EWSR1-ETS gene fusions. To identify secondary genetic lesions that contribute to tumor progression, we performed whole genome sequencing of 112 ES samples and matched germline DNA. Overall, ES tumors had relatively few single nucleotides variants, indels, structural variants and copy number alterations. Apart from whole chromosome arm copy number changes, the most common somatic mutations were in STAG2 (17%), CDKN2A (12%), TP53 (7%), EZH2, BCOR, ZMYM3 (2.7% each). Strikingly, STAG2 mutations and CDKN2A deletions were mutually exclusive, as confirmed in ES cell lines. In an expanded cohort of 299 patients with clinical data, we discovered that STAG2 and TP53 mutations are often concurrent and are associated with poor outcome. Finally, we detected sub-clonal STAG2-mutations in diagnostic tumors, and expansion of STAG2 immuno-negative cells in relapse as compared to matched diagnostic samples.