Precise detection of copy number aberrations (CNA) from tumor biopsies is critically important to the treatment of metastatic prostate cancer. The use of targeted panel next-generation sequencing (NGS) is inexpensive, high throughput, and easily feasible, allowing single-nucleotide variant calls, but CNA estimation from this remains challenging. We evaluated CNVkit for CNA identification from amplicon-based targeted NGS in a cohort of 110 fresh castration-resistant prostate cancer biopsies and used capture-based whole-exome sequencing (WES), array comparative genomic hybridization (aCGH), and FISH to explore the viability of this approach. We showed that this method produced highly reproducible CNA results ( = 0.92), with the use of pooled germline DNA as a coverage reference supporting precise CNA estimation. CNA estimates from targeted NGS were comparable with WES ( = 0.86) and aCGH ( = 0.7); for key selected genes (, and ), CNA estimation correlated well with WES ( = 0.91) and aCGH ( = 0.84) results. The frequency of CNAs in our population was comparable with that previously described (i.e., deep deletions: 4.5%; 8.2%; 15.5%; amplification: AR 45.5%; gain: MYC 31.8%). We also showed, utilizing FISH, that CNA estimation can be impacted by intratumor heterogeneity and demonstrated that tumor microdissection allows NGS to provide more precise CNA estimates. Targeted NGS and CNVkit-based analyses provide a robust, precise, high-throughput, and cost-effective method for CNA estimation for the delivery of more precise patient care. .