To provide effective molecular markers for the improvement of high temperature tolerance of Esox lucius, simplified genome sequencing was conducted based on the heat sensitive group (109) and high temperature tolerant group (103) of E. lucius. For InDel markers in 25 chromosomes, the first two PCA were covariables. The Masked Language Mode (MLM) was used to analyze the correlation between the heat tolerance characteristics of E. Lucius. The results showed that most InDel was distributed in introns (63.69%), while exons had few InDel sites (1.30%). Five loci were found to be significantly associated with heat tolerance traits (FDR P-value<0.05), which were located in the intron of safb gene, unknown gene LOC117593903 and CLSTN2 gene, respectively. The three InDel sites of 9N del, 4N del-1 and 4N del-2 were all located in the 3rd intron of Clstn2 gene, and the genotypes of the three sites were highly linked in 212 individuals. The five InDel mutations found in this study may have a significant effect on the heat tolerance traits of E. lucius, and can be used as candidate molecular markers for the improvement of heat tolerance traits of E. lucius. Further, some loci were verified by KASP technique in validation population. It was found that the 9N del site DD genotype was dominant in the heat sensitive group, and the DI genotype was dominant in the high temperature tolerance group, which was basically consistent with the simplified genome sequencing results. Three InDel sites located in the second intron of CLSTN2 gene may affect the transcription of CLSTN2 gene. This gene may be an important candidate gene related to high temperature tolerance in E. lucius. The results provide theoretical basis for marker assisted breeding and candidate molecular markers for the improvement of E. lucius heat tolerance traits.