Targeted next-generation sequencing (NGS) provides predictive and prognostic information in the routine care of patients with cancer. However, with increasing knowledge of the biological basis of cancer, NGS of the same gene sets can also provide diagnostic information in challenging cases, on the basis of identification of both known and novel variants, including single-nucleotide variants, insertions and deletions, copy number alterations, and translocations. Here, we present 3 clinical cases in which targeted NGS of hybrid-capture-enriched DNA from formalin-fixed, paraffin-embedded tumor samples provided unique and clinically important diagnostic and/or staging information in 3 different challenging clinical scenarios. In the first patient, NGS played a key role in both diagnosis and staging in a patient with multiple tumors of the same histologic type. The second case demonstrates the ability of NGS to clarify the tumor tissue type in a single mass involving multiple organs, and thereby guide appropriate chemotherapy. The third case illustrates that information regarding susceptibility to targeted therapeutics can also clarify the original histologic diagnosis.