Recurrent gene fusions between the androgen-regulated gene TMPRSS2 and the ETS family transcription factors ERG, ETV1, and ETV4 have been identified in the majority of prostate adenocarcinomas (PCA). PCA is often multifocal with histologic heterogeneity of different tumor foci. As TMPRSS2 is a common 5′ partner of ETS gene fusions, we monitored TMPRSS2 rearrangement by fluorescence in situ hybridization (FISH) to study the origin and molecular basis of multifocal PCA heterogeneity. TMPRSS2 rearrangement was evaluated by FISH on a tissue microarray representing 93 multifocal PCAs from 43 radical prostatectomy resections. Overall, 70% (30 of 43) of the cases showed TMPRSS2 rearrangement, including 63% through deletion (loss of the 3′ TMPRSS2 signal), 27% through translocation (split of 5′ and 3′ TMPRSS2 signals), and 10% through both mechanisms in different tumor foci. Of the 30 TMPRSS2 rearranged cases, 30% showed concordance in all tumor foci, whereas 70% were discordant in at least one focus. In TMPRSS2 rearranged cases, the largest (index) tumor was rearranged 83% of the time. Pathologic stage, size, or Gleason grade of the multifocal PCA did not correlate with overall TMPRSS2 rearrangement. Our results suggest that multifocal PCA is a heterogeneous group of diseases arising from multiple, independent clonal expansions. Understanding this molecular heterogeneity is critical to the future development and utility of diagnostic and prognostic PCA biomarkers. [Cancer Res 2007;67(17):7991–5]