Background: Sequential treatment with first-, second-generation ALK TKI followed by third-generation TKI (lorlatinib) have been widely applied to ALK-positive lung cancer. However, acquired resistance is often driven by secondary ALK mutations, which are needed to be further explored. Circulating tumor DNA, a non-invasive approach, can detect tumor-derived DNA from multiple metastatic sites and has become a promising strategy for assessing the genetic evolution of tumors and analyzing TKI resistance. Methods: Post-progression plasma specimens were collected and sequenced with a targeted gene panel from a total of 116 patients underwent ALK TKI treatment with from February 2014 to April 2020. The ALK TKIs administrated in this cohort included crizotinib (first-generation), ceritinib (second-generation), alectinib (second-generation), brigatinib (second-generation), ensartinib (second-generation) and lorlatinib (third-generation). Results: In this ALK-positive lung adenocarcinoma cohort, 49.1% were female and the rest were male with a median age of 52 (range 20 to 79). More than half of patients received more than one line of ALK TKI treatment. The most frequent ALK fusion type is EML4-ALK including EML4-ALK v1(E13; A20, 37/116, 31.9%) and EML4-ALKv3(E6a/b; A20, 49/116, 42.2%). TP53 (29.1%, 41/141) was the most frequently mutated gene other than ALK. Frequently identified secondary mutations in patients progressing on ALK inhibitors were ALK mutations L1196M and G1202R. ALK G1202R was more common in patient with v3 fusion than in v1 (25.8% vs 0%; P<.001) while ALK L1196M was more common in v1 than in v3 (20.8% vs 3.2%; P=.005). Meanwhile, G1202R was identified at higher ratio in patients progressed on second-generation ALK TKI than first- and third-generation ALK TKI (11.3% vs 0% and 9.1%) whereas L1196M was more found in patients progressed on first-generation ALK TKI (9.1% vs 1.9% and 4.5%). Other identified secondary mutations were ALK F1174C/V/L, E1210K/Q, D1269A, D1203N and L1122M/V. Compound ALK mutations (≥2 concurrent ALK mutations) were more common in patients relapsed on third-generation ALK TKI lorlatinib (36.4%) compared to first- (12.1%) and second-generation (20.8%) ALK TKIs. Using serial plasma specimens, clone evolution analysis showed that five patients developed compound ALK mutations after sequential ALK TKI treatments and two novel ALK compound lorlatinib-resistant mutations D1203N+I1171N and F1174C+G1202R were identified. Conclusions: Genotyping of sequential post-progression plasma specimens reveals that treatment with sequential first-, second-, and third-generation ALK inhibitors can accelerate the accumulation of ALK resistance mutations and may lead to treatment-refractory compound ALK mutations. The selection for optimal first-line TKI is very important to achieve a more efficacious long-term strategy and prevent the emergence of on-target resistance.