High temperature, which can reduce crop seed vigor, slow down or completely inhibit germination, is one of the important abiotic stresses that restrict cucumber production. A set of core germplasm including 96 cucumber accessions were used to identify thermotolerance at high temperature 42℃ during the seed germination stage. The thermotolerance was evaluated using a comprehensive index, which include the relative germination percentage, relative germination vigor, relative germination index and relative radicle length. The re-sequencing data of the core germplasm were used to perform the Genome-wide association study (GWAS). We identified two accessions, CG109 and CG110, which have a pretty strong thermotolerance, with the comprehensive index of over 0.9. We detected 5 loci for relative germination percentage: GGP4.1, GGP4.2, GGP5.1, GGP5.2 and GGP6.1; 2 for relative germination vigor: GGV4.1 and GGV5.1; 2 for relative germination index: GGI4.1 and GGI5.1; 2 for relative radicle length: GRL6.1 and GRL7.1. Among them, GGI4.1, GGV4.1 and GGP4.1 are located to the same physical region of Chr.4, and GGI5.1, GGV5.1 and GGP5.2 are located to the same region of Chr.5. The loci located in these two regions can be detected simultaneously by relative germination percentage, relative germination vigor and relative germination index three different indicators, which suggested that the two regions could include genes related to thermotolerance.