To characterize the clonality of TMPRSS2-ERG fusion in multifocal prostate cancer.From 80 consecutive radical prostatectomy specimens, we identified 32 cases with multiple spatially separate tumors. In each case, we assessed two to three tumor foci for TMPRSS2-ERG fusion using an ERG break-apart interphase fluorescence in situ hybridization assay.Individual tumor foci showed homogeneity for fusion status (intrafocal clonal homogeneity). In 19 (59\%) of the 32 cases, all foci within a case had the same fusion status (interfocal homogeneity). In 15 (80\%) of the 19 cases, no foci had fusion, and in 4 (20\%), all foci had fusion. Of the 32 cases, 13 (41\%) demonstrated heterogeneity for fusion status within a case (interfocal clonal heterogeneity).In this study, we have demonstrated interfocal heterogeneity and intrafocal homogeneity for TMPRSS2-ERG fusion in prostate cancer with multiple tumors. These findings support the multiclonal nature of prostate cancer with clinical implications for needle biopsy strategies and the development of urine-based screening tests.