Background: Using targeted ultradeep sequencing, we aimed to explore genomic alterations that confer extreme sensitivity to everolimus. Methods: We collected formalin-fixed paraffin-embedded tumor/normal pairs from 38 patients (21 with exceptional clinical benefit, 17 with no clinical benefit) who were treated with everolimus across various tumor types (13 gastric cancers, 15 renal cell carcinomas, 2 thyroid cancers, 1 lacrimal gland cancer, and 7 sarcomas). Ion AmpliSeqComprehensive Cancer Panel was used to identify alterations across all exons of 409 target genes. Results: Tumors were sequenced to a median coverage of 605x. Cancer genomes are characterized by 203 somatic single-nucleotide variants (186 missense, 10 nonsense, 7 splice-site) and 51 frameshift insertions/deletions, with a median of 3.9 mutations per Mb (0 to 14.8 mutations per Mb). Overall, genomic alterations with activating effect on mTOR signaling were identified in 8 of 21 patients with clinical benefit and these include mTOR, TSC1, TSC2, NF1 and PIK3CA mutations. In a patient with ductal adenocarcinoma of the lacrimal gland in whom everolimus achieved a partial response (PR) for 8 months, a novel NF1 missense mutation (D1644A) and a novel TP53 frameshift deletion (A39fs*5) were revealed. Three mTOR missense mutations (K1771R, N1421D, I1973F) were found in patients with gastric cancer, renal cell carcinoma and angiosarcoma. A mutation in the helical domain of PIK3CA (E542K) was found in a renal cell carcinoma patient and a mutation in AKT1 (H238Y) was observed in a sarcoma patient with malignant fibrous histiocytoma. Lastly, a patient with anaplastic thyroid cancer harbored a nonsense mutation in TSC1 (p.Trp103*) and a renal cell carcinoma patient had a TSC1 splicing variant (c.1029+1G > A). Recurrent mutations in chromatin remodeling gene (BAP1; n = 2, 12%)and receptor tyrosine kinase signaling (FGFR4; n = 2, 12%) were noted only in patients without clinical benefit. Conclusions: Regardless of different cancer types, mTOR-pathway-activating mutations confer sensitivity to everolimus. Targeted sequencing of mTOR pathway genes facilitates identification of potential candidates for mTOR inhibitors.