Lorlatinib is a potent, brain-penetrant, third-generation anaplastic lymphoma kinase (ALK)/ROS1 tyrosine kinase inhibitor (TKI) with robust clinical activity in advanced ALK-positive non-small-cell lung cancer, including in patients who have failed prior ALK TKIs. Molecular determinants of response to lorlatinib have not been established, but preclinical data suggest that resistance mutations may represent a biomarker of response in previously treated patients. Baseline plasma and tumor tissue samples were collected from 198 patients with ALK-positive non-small-cell lung cancer from the registrational phase II study of lorlatinib. We analyzed plasma DNA for mutations using Guardant360. Tumor tissue DNA was analyzed using an mutation-focused next-generation sequencing assay. Objective response rate, duration of response, and progression-free survival were evaluated according to mutation status. Approximately one quarter of patients had mutations detected by plasma or tissue genotyping. In patients with crizotinib-resistant disease, the efficacy of lorlatinib was comparable among patients with and without mutations using plasma or tissue genotyping. In contrast, in patients who had failed 1 or more second-generation ALK TKIs, objective response rate was higher among patients with mutations (62% 32% plasma; 69% 27% tissue). Progression-free survival was similar in patients with and without mutations on the basis of plasma genotyping (median, 7.3 months 5.5 months; hazard ratio, 0.81) but significantly longer in patients with mutations identified by tissue genotyping (median, 11.0 months 5.4 months; hazard ratio, 0.47). In patients who have failed 1 or more second-generation ALK TKIs, lorlatinib shows greater efficacy in patients with mutations compared with patients without mutations. Tumor genotyping for mutations after failure of a second-generation TKI may identify patients who are more likely to derive clinical benefit from lorlatinib.