Abstract: Objective The anaplastic lymphoma kinase （ALK） fusion was an important driver gene of non-small-cell lung cancer （NSCLC）, and ALK-tyrosine kinase inhibitor （TKI） have significantly improved clinical outcomes in patients with ALK fusion. Unfortunately, resistance to ALK-TKI inevitably occurs after a period of treatment. The main mechanisms of resistance have been found to include ALK secondary resistance mutations, ALK gene amplification, bypass and downstream pathway activation. The aim of this study was to investigate the mechanism of resistance to crizotinib in clinical practice. Methods We retrospectively collected a total of 234 patients with ALK-positive advanced NSCLC from January 2016 to December 2020 at Guangdong Lung Cancer Institute. Ventana immunohistochemistry （IHC）, fluorescence in situ hybridization （FISH）, tissue or liquid next-generation sequencing （NGS） were conducted to confirm ALK rearrangement. Tissues / plasma based NGS was performed again after ALK-TKI resistance. Clinicopathological and molecular characteristics and clinical data were collected for Kaplan Meier analysis. Results We screened a total of 44 patients with advanced ALK-rearranged NSCLC treated with crizotinib and who underwent at least 1 repeat biopsy （including 31 plasma specimens and 39 tissue specimens） after disease progression. Echinoderm microtubule associated protein like 4-anaplastic lymphoma kinase （EML4-ALK） variant 3 （v3） was the most common ALK variant in this cohort, followed by EML4-ALK variant 1 （v1）. Of these variants, v3 cohort was more likely to developed ALK G1202R resistance mutation after crizotinib resistance compared to the other variants （0% vs. 38%, P=0.006 4）. A second site mutation resistance mechanism in the ALK tyrosine kinase region was identified in 21 of these 44 patients （48%）, with L1196M accounting for the most （13%）, followed by more than 2 ALK mutations （11%） and G1202R （7%）. Survival analysis showed that the progression-free survival （PFS） of v3 and other variants groups treated with crizotinib were both 8.8 months, with no statistically significant difference （P=0.683 6）. Survival analysis based on the presence or absence of ALK resistance mutation after drug resistance showed that PFS was 6.6 months in the ALK resistance mutation group, and 8.8 months in the absence of ALK resistance mutation group, with no statistically significant difference （P=0.615 0）. Conclusions ALK kinase domain mutations predominate after crizotinib resistance in patients with advanced ALK rearranged NSCLC. EML4-ALK v3 variants were more likely to produce ALK G1202R resistance mutations after crizotinib resistance.