In this study, the core collection of cucumber germplasm accessions were used to evaluate low-temperature tolerance at seedling stage, and the candidate genes were identified via genome-wide association analysis. Two-mature-leaf seedling plants were exposed under low temperature conditions (12℃ or 19.3℃) for 14 days and 11 days, respectively. The classification of low-temperature tolerance was conducted based on the chilling injury index (CII) of cotyledons and true leaves. After treatments, the difference of seedlings was significant, and the coefficient of variation under two environmental conditions were 23.2% and 31.7%, respectively. These accessions were divided into four groups according to CII. Four accessions (CG45, CG61, CG88 and CG104) were shown to be tolerant under low-temperature stress. The genome-wide association mapping of low temperature tolerance identified four loci including gLTS1.1, gLTS3.1, gLTS4.1 and gLTS5.1 that were assigned to the chromosomes 1, 3, 4 and 5 respectively. Among them, the locus gLTS5.1 can be detected repeatedly. Thus, this work provided insight for identifying low-temperature tolerant cucumber germplasm and unlocking the functional genes in future.