Low temperatures (LTs) negatively affect the percentage and rate of cucumber (Cucumis sativusL.) seed germination, which has deleterious effects on yield. Here, a genome-wide association study (GWAS) was used to identify the genetic loci underlying low temperature germination (LTG) in 151 cucumber accessions that represented seven diverse ecotypes. Over two years, phenotypic data for LTG i.e., relative germination rate (RGR), relative germination energy (RGE), relative germination index (RGI) and relative radical length (RRL), were collected in two environments, and 17 of the 151 accessions were found to be highly cold tolerant using cluster analysis. A total of 1,522,847 significantly associated single-nucleotide polymorphism (SNP) were identified, and seven loci associated with LTG, on four chromosomes, were detected:gLTG1.1,gLTG1.2,gLTG1.3,gLTG4.1,gLTG5.1,gLTG5.2, andgLTG6.1after resequencing of the accessions. Of the seven loci, three, i.e.,gLTG1.2,gLTG4.1, andgLTG5.2, showed strong signals that were consistent over two years using the four germination indices, and are thus strong and stable for LTG. Eight candidate genes associated with abiotic stress were identified, and three of them were potentially causal to LTG:CsaV3_1G044080(a pentatricopeptide repeat-containing protein) forgLTG1.2,CsaV3_4G013480(a RING-type E3 ubiquitin transferase)for gLTG4.1, andCsaV3_5G029350(a serine/threonine-protein kinase) forgLTG5.2.The function forCsPPR(CsaV3_1G044080) in regulating LTG was confirmed, asArabidopsislines ectopically expressingCsPPRshowed higher germination and survival rates at 4°C compared to the wild-type, which preliminarily illustrates thatCsPPRpositively regulates cucumber cold tolerance at the germination stage. This study will provide insights into cucumber LT-tolerance mechanisms and further promote cucumber breeding development.