To characterize the pattern of ETS rearrangements and to uncover novel ETS fusion genes, we analyzed 200 prostate carcinomas (PCa) with TaqMan low-density arrays (TLDAs), followed by selective analyses with fluorescence in situ hybridization (FISH), RT-PCR, and sequencing. Besides confirming the recurrent presence ofERG,ETV1,ETV4, andETV5rearrangements, we here reportFLI1as the fifth ETS transcription factor involved in fusion genes in prostate cancer. Outlier expression of theFLI1gene was detected by TLDAs in one PCa that showed relative overexpression ofFLI1exons 4:5 as compared withFLI1exons 2:3. A structural rearrangement was found using FISH probes flanking theFLI1gene and RT-PCR and sequencing analyses showed fusion ofSLC45A3exon 1 withFLI1exon 3. Interestingly, we found four cases with two different ETS rearrangements in the index tumor, thus revealing intratumor genetic heterogeneity. Correlation analysis with clinico-pathological data showed association ofERGrearrangements with locally advanced disease (pT3,P= 0.007) andMYCoverexpression (P= 0.001), and association ofETV1rearrangements withPTENdownregulation (P= 0.015). We report thatFLI1is a novel ETS transcription factor involved in gene fusions in prostate cancer and that intratumor genetic heterogeneity of ETS rearrangements can occasionally be found in index primary tumors.