In the phase III TH3RESA study (NCT01419197), 602 patients with HER2‐positive advanced breast cancer who received prior taxane therapy and ≥2 HER2‐directed regimens, including trastuzumab and lapatinib (advanced setting), were randomized to trastuzumab emtansine (T‐DM1) or treatment of physician's choice (TPC). A statistically significant progression‐free survival (PFS) benefit favoring T‐DM1 was demonstrated. Here, we examine the relationship between HER2‐related biomarkers and PFS in an exploratory analysis. Biomarkers assessed included HER2 (n = 505) and HER3 (n = 505) mRNA expression, PIK3CA mutation status (n = 410) and PTEN protein expression (n = 358). For biomarkers with continuous data (HER2, HER3, PTEN), subgroups were defined using median values (>median and ≤median). For all biomarker subgroups, median PFS was longer with T‐DM1 vs. TPC. The PFS benefit favoring T‐DM1 vs. TPC was numerically greater in the HER2 mRNA >median subgroup (7.2 vs. 3.4 months; unstratified hazard ratio [HR], 0.40; 95% CI, 0.28–0.59; p < 0.0001) vs. ≤median subgroup (5.5 vs. 3.9 months; HR, 0.68; 95% CI, 0.49–0.92; p = 0.0131). The PFS benefit with T‐DM1 was similar among HER3, PIK3CA and PTEN subgroups. Consistent with other reports, benefit was seen with T‐DM1 regardless of PIK3CA mutation status. In a multivariate analysis including an interaction term (treatment group by log2‐transformed HER2 mRNA), patients with higher HER2 mRNA levels benefited more from receiving T‐DM1 (HR, 0.84; 95% CI, 0.75–0.94; interaction p value = 0.0027). In summary, T‐DM1 prolonged median PFS in all biomarker subgroups analyzed, including activating PIK3CA mutations, with numerically greater benefit in patients with tumors expressing HER2 mRNA >median vs. ≤median.