Most Ewing family tumors are identified by the characteristic translocation t(11;22)(q24;q12), resulting in a fusion protein EWS/FLI1 that acts as an aberrant transcription factor. In a minority of cases, the EWS gene is fused to another member of the ETS gene (ERG, ETV1, E1AF, and FEV). Though the oncogenic transforming capability of the EWS/FLI1 protein is highly suggestive, the exact pathway behind remains to be elucidated. The availability of cell lines may help in the understanding of underlying cellular processes. In this study, we have established two new Ewing sarcoma cell lines and characterized them with molecular cytogenetic tools. This technology was also applied on four other previously published Ewing sarcoma cell lines. Our findings in relation to previous data on similar tumors are discussed.