BACKGROUND Most personalized cancer care strategies involving DNA sequencing are highly reliant on acquiring sufficient fresh or frozen tissue. It has been challenging to comprehensively evaluate the genome of advanced prostate cancer (PCa) because of limited access to metastatic tissue. OBJECTIVE To demonstrate the feasibility of a novel next-generation sequencing (NGS)-based platform that can be used with archival formalin-fixed paraffin-embedded (FFPE) biopsy tissue to evaluate the spectrum of DNA alterations seen in advanced PCa. DESIGN, SETTING, AND PARTICIPANTS FFPE samples (including archival prostatectomies and prostate needle biopsies) were obtained from 45 patients representing the spectrum of disease: localized PCa, metastatic hormone-naive PCa, and metastatic castration-resistant PCa (CRPC). We also assessed paired primaries and metastases to understand disease heterogeneity and disease progression. INTERVENTION At least 50 ng of tumor DNA was extracted from FFPE samples and used for hybridization capture and NGS using the Illumina HiSeq 2000 platform. OUTCOME MEASUREMENTS AND STATISTICAL ANALYSIS A total of 3320 exons of 182 cancer-associated genes and 37 introns of 14 commonly rearranged genes were evaluated for genomic alterations. RESULTS AND LIMITATIONS We obtained an average sequencing depth of >900X. Overall, 44% of CRPCs harbored genomic alterations involving the androgen receptor gene (AR), including AR copy number gain (24% of CRPCs) or AR point mutation (20% of CRPCs). Other recurrent mutations included transmembrane protease, serine 2 gene (TMPRSS2):v-ets erythroblastosis virus E26 oncogene homolog (avian) gene (ERG) fusion (44%); phosphatase and tensin homolog gene (PTEN) loss (44%); tumor protein p53 gene (TP53) mutation (40%); retinoblastoma gene (RB) loss (28%); v-myc myelocytomatosis viral oncogene homolog (avian) gene (MYC) gain (12%); and phosphatidylinositol-4,5-bisphosphate 3-kinase, catalytic subunit α gene (PIK3CA) mutation (4%). There was a high incidence of genomic alterations involving key genes important for DNA repair, including breast cancer 2, early onset gene (BRCA2) loss (12%) and ataxia telangiectasia mutated gene (ATM) mutations (8%); these alterations are potentially targetable with poly(adenosine diphosphate-ribose)polymerase inhibitors. A novel and actionable rearrangement involving the v-raf murine sarcoma viral oncogene homolog B1 gene (BRAF) was also detected. CONCLUSIONS This first-in-principle study demonstrates the feasibility of performing in-depth DNA analyses using FFPE tissue and brings new insight toward understanding the genomic landscape within advanced PCa.