To investigate the cucumber thermotolerance at seedling stage and identify heat-tolerant accessions, this study analyzed 86 cucumber core germplasm at three-mature-leaves seedlings stage under greenhouse conditions (temperature: 50±4℃). Scoring for heat damage was conducted based on heat injury symptoms, and the heat injury index (HII) was deployed to evaluate the thermotolerance of cucumber at seedling stage. Taking advantage of the whole genome re-sequencing datasets, the genome-wide association study (GWAS) was employed to identify the genetic loci that associated to the thermotolerance. The significant differences in HII were detected among tested materials, while the coefficient of variation under two environmental conditions were 21.9% and 22.5% respectively. By deploying HII, 86 cucumber germplasms were clustered into four groups: heat-resistant, medium heat-resistant, medium heat-sensitive and heat-sensitive group. The preliminary GWAS analysis suggested seven loci, including gHII4.1、gHII5.1、gHII5.2、gHII6.1、gHII7.1、gHII4.2 and gHII6.2, which were associated to the heat resistance. Among them, two loci gHII4.1 and gHII4.2 on chromosome 4, were found to be mostly relevant, and 67 candidate genes within this region were found.