Acquired resistance to next-generation ALK tyrosine kinase inhibitors (TKIs) is often driven by secondary mutations. Here, we investigated utility of plasma genotyping for identifying resistance mutations at relapse on next-generation ALK TKIs. We analyzed 106 plasma specimens from 84 patients with advanced -positive lung cancer treated with second- and third-generation ALK TKIs using a commercially available next-generation sequencing (NGS) platform (Guardant360). Tumor biopsies from TKI-resistant lesions underwent targeted NGS to identify mutations. By genotyping plasma, we detected an mutation in 46 (66%) of 70 patients relapsing on a second-generation ALK TKI. When post-alectinib plasma and tumor specimens were compared, there was no difference in frequency of mutations (67% vs. 63%), but plasma specimens were more likely to harbor ≥2 mutations (24% vs. 2%, = 0.004). Among 29 patients relapsing on lorlatinib, plasma genotyping detected an mutation in 22 (76%), including 14 (48%) with ≥2 mutations. The most frequent combinations of mutations were G1202R/L1196M and D1203N/1171N. Detection of ≥2 mutations was significantly more common in patients relapsing on lorlatinib compared with second-generation ALK TKIs (48% vs. 23%, = 0.017). Among 15 patients who received lorlatinib after a second-generation TKI, serial plasma analysis demonstrated that eight (53%) acquired ≥1 new mutations on lorlatinib. resistance mutations increase with each successive generation of ALK TKI and may be underestimated by tumor genotyping. Sequential treatment with increasingly potent ALK TKIs may promote acquisition of resistance mutations leading to treatment-refractory compound mutations.