To explore whether the progression-free survival (PFS) with pemetrexed differs between anaplastic lymphoma kinase (ALK)-positive and other major molecular subtypes of non-small cell lung cancer.In an ALK-enriched population, patients with metastatic non-small cell lung cancer were screened by ALK fluorescence in situ hybridization and for epidermal growth factor receptor (EGFR) and KRAS mutations. Triple-tested, pemetrexed-treated patients (monotherapy or combination therapy) were identified and PFS with pemetrexed captured retrospectively.Eighty-nine eligible cases were identified (19 ALK fluorescence in situ hybridization positive, 12 EGFR mutant, 21 KRAS mutant, and 37 triple negatives). Eighty-three cases (93%) were adenocarcinomas, two were adenosquamous, one squamous, and three had large cell histology. None of the ALK-positive patients had received crizotinib before pemetrexed. Pemetrexed was first-line therapy in 48% (72% as platinum-based combinations). Median PFS (95% confidence interval) data were EGFR mutant (5.5 months; 1-9), KRAS mutant (7 months; 1.5-10), ALK positive (9 months; 3-12), and triple negative (4 months; 3-5). In a multivariate analysis adjusting for line of therapy, mono- versus platinum and nonplatinum combination therapy, age, sex, histology, and smoking status, the only variable associated with prolonged PFS on pemetrexed was ALK+ (hazard ratio = 0.36 [95% confidence interval: 0.17-0.73], p = 0.0051).In this exploratory analysis, ALK-positive patients have a significantly longer PFS on pemetrexed compared with triple-negative patients, whereas EGFR or KRAS mutant patients do not. This information should be considered when sizing randomized studies in ALK-positive patients that involve pemetrexed. Pemetrexed should also be prioritized as a cytotoxic to explore further in patients with known ALK-positive disease.