Background: Next-generation ALK tyrosine kinase inhibitors (TKIs) are the cornerstone of management of ALK-positive (ALK+) lung cancer. Each ALK TKI has a unique spectrum of activity against distinct ALK kinase domain mutations (muts). Plasma genotyping is a promising strategy for identifying ALK muts at relapse on ALK TKIs. Methods: To detect ALK muts, we performed next-generation sequencing (Guardant360) of circulating tumor DNA from patients (pts) with ALK+ lung cancer relapsing on a second-generation (2^nd-gen) ALK TKI (n = 65) or the third-generation (3^rd-gen) TKI lorlatinib (n = 26). Results: Among 65 pts progressing on a 2^nd-gen TKI, 49 (75%) had only received one 2^nd-gen ALK TKI prior to analysis: n = 42 alectinib, n = 3 each brigatinib/ceritinib, and n = 1 ensartinib. We detected an ALK mut in 42/65 (65%) specimens at relapse, among which ALK G1202R (32%) and I1171X (23%) were the most common. Sixteen (25%) pts had ≥2 ALK muts at progression on a 2^nd-gen TKI. Among 26 pts progressing on lorlatinib (all of whom had previously relapsed on a 2^nd-gen ALK TKI), we identified ALK muts in 20 (76%), including 14 (54%) with ≥2 ALK muts. Detection of ≥2 ALK muts was more common at relapse on lorlatinib compared to a 2^nd-gen TKI (p = 0.013). To assess the evolution of ALK muts during treatment with different TKIs, we analyzed serial plasma specimens from 20 pts treated with sequential 2^nd-gen/2^nd-gen or 2^nd-gen/3^rd-gen TKIs. Among six pts who received alectinib followed by brigatinib, repeat plasma analysis at brigatinib progression revealed persistence of pre-brigatinib ALK muts in two pts (one L1196M and one G1202R), expansion of G1202R in one pt, and acquisition of new ALK muts in three pts. Among 14 pts who received a 2^nd-gen TKI followed by lorlatinib, 11 had persistence of pre-lorlatinib ALK muts and 8 acquired ≥1 additional ALK muts at lorlatinib progression. The most frequently acquired ALK mut was D1203N in four of eight cases. Conclusions:ALK resistance muts are prevalent at relapse on next-generation ALK TKIs and increase with each successive generation of ALK TKIs. These findings suggest that sequential therapy with increasingly potent ALK TKIs may select for compound ALK muts and/or fuel tumor heterogeneity.