Two recent phase III trials, BOLERO-1 and BOLERO-3 (Breast Cancer Trials of Oral Everolimus), evaluated the addition of everolimus to trastuzumab and chemotherapy in human epidermal growth factor receptor 2-overexpressing advanced breast cancer. The current analysis aimed to identify biomarkers to predict the clinical efficacy of everolimus treatment. Archival tumor samples from patients in BOLERO-1 and BOLERO-3 were analyzed using next-generation sequencing, immunohistochemistry, and Sanger sequencing. Biomarker data were available for 549 patients. PIK3CA activating mutations and PTEN loss were reported in 30% and 16% of BOLERO-1 samples and in 32% and 12% of BOLERO-3 samples, respectively. PI3K pathway was hyperactive (PIK3CA mutations and/or PTEN loss and/or AKT1 mutation) in 47% of BOLERO-1 and 41% of BOLERO-3 samples. In both studies, differential progression-free survival (PFS) benefits of everolimus were consistently observed in patient subgroups defined by their PI3K pathway status. When analyzing combined data sets of both studies, everolimus was associated with a decreased hazard of progression in patients with PIK3CA mutations (hazard ratio HR, 0.67; 95% CI, 0.45 to 1.00), PTEN loss (HR, 0.54; 95% CI, 0.31 to 0.96), or hyperactive PI3K pathway (HR, 0.67; 95% CI, 0.48 to 0.93). Patients with wild-type PIK3CA (HR, 1.10; 95% CI, 0.83 to 1.46), normal PTEN (HR, 1.00; 95% CI, 0.80 to 1.26), or normal PI3K pathway activity (HR, 1.19; 95% CI, 0.87 to 1.62) did not derive PFS benefit from everolimus. This analysis, although exploratory, suggests that patients with human epidermal growth factor receptor 2-positive advanced breast cancer having tumors with PIK3CA mutations, PTEN loss, or hyperactive PI3K pathway could derive PFS benefit from everolimus.