Purpose To explore the genetic landscape of tumors from patients enrolled in the BOLERO-2 trial to identify potential correlations between genetic alterations and efficacy of everolimus treatment. The BOLERO-2 trial has previously demonstrated that adding everolimus to exemestane prolonged progression-free survival (PFS) by greater than twofold in patients with hormone receptor-positive, human epidermal growth factor receptor-2–negative advanced breast cancer previously treated with nonsteroidal aromatase inhibitors. Methods Next-generation sequencing was used to analyze genetic status of cancer-related genes in 302 archival tumor specimens from patients representative of the BOLERO-2 study population. Correlations between the most common somatic alterations and degree of chromosomal instability, and treatment effect of everolimus were investigated. Results Progression-free survival benefit with everolimus was maintained regardless of alteration status of PIK3CA, CCND1, and TP53 genes or the pathways of which they are components. However, quantitative differences in everolimus benefit were observed between patient subgroups defined by the mutation locations in PIK3CA (exon 20 and other exons). In addition, the degree of chromosomal instability correlated with treatment efficacy in the everolimus arm. Conclusion The data suggest that adding everolimus to exemestane results in marked prolongation of PFS in postmenopausal women with hormone receptor-positive breast cancer whose tumors have limited chromosomal instabilities and abnormalities in potentially compensatory signaling pathways controlling growth/survival, cell-cycle progression, or DNA damage/repair. (Funded by Novartis; BOLERO-2 ClinicalTrials.gov number, NCT00863655.)