Improvement of persistency is an important breeding goal in red clover (Trifolium pratenseL.). In areas with cold winters, lack of persistency is often due to poor winter survival, of which low freezing tolerance (FT) is an important component. We conducted a genome wide association study (GWAS) to identify loci associated with freezing tolerance in a collection of 393 red clover accessions, mostly of European origin, and performed analyses of linkage disequilibrium and inbreeding. Accessions were genotyped as pools of individuals using genotyping-by-sequencing (pool-GBS), generating both single nucleotide polymorphism (SNP) and haplotype allele frequency data at accession level. Linkage disequilibrium was determined as a squared partial correlation between the allele frequencies of pairs of SNPs and found to decay at extremely short distances (< 1 kb). The level of inbreeding, inferred from the diagonal elements of a genomic relationship matrix, varied considerably between different groups of accessions, with the strongest inbreeding found among ecotypes from Iberia and Great Britain, and the least found among landraces. Considerable variation in FT was found, with LT50-values (temperature at which 50% of the plants are killed) ranging from -6.0°C to -11.5°C. SNP and haplotype-based GWAS identified eight and six loci significantly associated with FT (of which only one was shared), explaining 30% and 26% of the phenotypic variation, respectively. Ten of the loci were found within or at a short distance (<0.5 kb) from genes possibly involved in mechanisms affecting FT. These include a caffeoyl shikimate esterase, an inositol transporter, and other genes involved in signaling, transport, lignin synthesis and amino acid or carbohydrate metabolism. This study paves the way for a better understanding of the genetic control of FT and for the development of molecular tools for the improvement of this trait in red clover through genomics assisted breeding.