Abstract Ewing sarcoma is typically a bone tumor of adolescence characterized by a small round cell phenotype, CD99 membrane staining and presence of a gene fusion between FET family members (most frequently EWSR1, much more rarely FUS/TLS or TAF15) and members of the ETS family of transcription factors (mainly FLI1 or ERG, more rarely others). A first diversity relates to the type of fusion. Indeed, recently, other types of gene fusions have been observed in tumors suspected to be Ewing sarcoma but lacking the typical gene fusion. These tumors have been collectively named Ewing-like sarcoma. They include tumors with the CIC-DUX4, EWSR1-NFATc2, BCOR-CCNB3 and EWSR1-PATZ1 fusions. To more precisely investigate the relationships between these different tumors we first screened a large tumor bank of bone and soft tissue sarcoma for these different fusions then profiled the gene expression of a set of 117 FET-ETS, 12 BCOR-CCNB3, 12 CIC-DUX4, 8 EWSR1-NFATc and 5 EWSR1-PATZ1 cases. Results strongly suggest that there is strong homogeneity within each fusion categories but that the 5 types of fusion define 5 different biological tumor entities. In addition, when the clinical characteristics of these tumors are investigated some important differences are observed. They mostly concern the age of onset and the primary localization of the tumor. A second type of diversity concerns secondary genetic alterations. In collaboration with the Pediatric Cancer Genome Project at St Jude we studied a series of 112 Ewing sarcomas by whole genome sequencing comparing germline and tumor DNA. Overall, Ewing sarcoma tumors had relatively few single-nucleotide variants, indels, structural variants, and copy-number alterations. Apart from whole chromosome arm copy-number changes, the most common somatic mutations were detected in STAG2 (17%), CDKN2A (12%), TP53 (7%), EZH2, BCOR, and ZMYM3 (2.7% each). Strikingly, STAG2 mutations and CDKN2A deletions were mutually exclusive, as confirmed in Ewing sarcoma 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 subclonal STAG2 mutations in diagnostic tumors and expansion of STAG2-immunonegative cells in relapsed tumors as compared with matched diagnostic samples. Another level of diversity is the cell-to-cell phenotypic heterogeneity within a population of cells with the same genetic background. Indeed, different types of investigations including FACS, single cell RNA seq and functional analyses suggest that non-genetic heterogeneity plays a crucial role in a variety of tumor related processes including migration, resistance and ability to seed in distant organs. Citation Format: Olivier Delattre. Genetic and phenotypic diversity in Ewing sarcoma. [abstract]. In: Proceedings of the AACR Special Conference on Advances in Pediatric Cancer Research: From Mechanisms and Models to Treatment and Survivorship; 2015 Nov 9-12; Fort Lauderdale, FL. Philadelphia (PA): AACR; Cancer Res 2016;76(5 Suppl):Abstract nr IA15.