Ewing's sarcoma family of tumors (ESFTs) are indicated by malignant, small, round and blue cell tumors of the bone and soft tissue. Gene rearrangements between EWS gene on chromosome 22q12 and members of the ETS gene family are common in and specific to ESFTs. Another defining characteristic of ESFTs is their membranous expression of the CD99. In contrast, such translocations and immunoreactivity are not found in central primitive neuroectodermal tumors (cPNETs). The aim of this study was to investigate the detection of EWS/FLI1 translocations and CD99 immunoreactivity in order to evaluate their clinicopathological features and their roles in the differential diagnosis of these tumors. In this study, we investigated CD99 immunoreactivity using immunohistochemistry and Ewing's sarcoma / Friend leukaemia virus integration 1 (EWS/FLI1) translocation using the fluorescence in situ hybridization (FISH) method in 23 cases. CD99 expression was detected in 10/11 (90%) ESFT cases and 2/7 cPNET cases. In 18 cases EWS/FLI1 translocation was examined using the FISH method. The EWS/FLI1 translocations were detected in 7/8 (87.5%) ESFTs cases, whereas non of 8 cPNET cases were detected with this translocation. One case could not be classified as either central or peripheral, showed EWS/FLI1 translocation. There was a statistically significant difference in CD99 expression (p = 0.0013) and EWS/FLI1 translocation (p = 0,002) between cPNETs and ESFTs cases. In conclusion, CD99 expression and EWS/FLI1 translocation are specific and sensitive markers in the diagnosis of ESFTs. However, these were often not found in cases of cPNET. Therefore, in the diagnosis of ESFTs, clinical, radiological, histopathological and immunohistochemical parameters should always be evaluated together.