TMPRSS2 gene fusions with ETS transcription factor family members ERG, ETV1, or ETV4 have been recently discovered as a common molecular event in prostate cancer. Much attention has been focused on exploring their clinical application as a genetic tumor marker for the diagnosis, prognosis, and prediction of response to therapy. Although several studies have been done, the clinical utility of TMPRSS2 genetic alterations as biomarkers for prostate carcinoma remains indeterminate. In this study, we examined adenocarcinomas, prostatic intraepithelial neoplasia (PIN), and normal epithelium of the prostate retrieved from radical prostatectomy specimens to determine the frequency, specificity, tissue heterogeneity, and prognostic value of TMPRSS2 genetic alterations using a direct-labeled TMPRSS2 dual-color break-apart fluorescence in situ hybridization (FISH) probe cocktail designed to detect all known TMPRSS2-associated deletions or translocations. Seventy-one patients (161 samples) with normal prostate tissue, 60 patients (153 samples) with PIN, and 61 patients (142 samples) with carcinoma in formalin-fixed paraffin-embedded tissue microarrays were tested. None of the 161 normal prostate samples showed TMPRSS2 translocation or deletion. Sixty-two percent patients of prostate carcinomas demonstrated TMPRSS2 gene alterations, including 39% with translocation, 16% with deletion, and 7% with a mixed pattern. Tissue heterogeneity for TMPRSS2 gene alterations was identified in 28% of prostate carcinomas. No difference in the frequency of TMPRSS2 gene alterations was found between Gleason 6 and 7 tumors. Seventeen percent of PIN had TMPRSS2 gene alterations and showed the same FISH patterns as in the carcinomas from respective prostatectomy specimens. The TMPRSS2 dual-color break-apart FISH probe cocktail provides a simple and reliable method for the detection of TMPRSS2-related genetic alterations in formalin-fixed paraffin-embedded tissue. TMPRSS2 genetic alterations detectable by this method are strictly restricted in prostate neoplasia, and can be identified in the majority of prostate carcinomas. Tissue heterogeneity for TMPRSS2 alterations is common, and it should be considered when sampling and evaluating biopsy specimens.