Perennial ryegrass (Lolium perenne L.) is considered a premier turfgrass and forage grass worldwide yet susceptibility to freezing damage limits its use. C-repeat binding factor (CBF) dehydration-responsive element-binding protein (DREB) related genes are considered important for the regulation of abiotic stress in plants. In this study, CBF DREB-related genes in perennial ryegrass, also known as LpCBF genes, were resequenced from cultivated and landrace plants from a worldwide collection. A total of 109 plants survived clonal propagation and were tested for freezing tolerance. The resulting data was subjected to mixed model analysis to determine whether the freezing tolerance is related to single nucleotide polymorphism (SNP) and indel markers within two genes, LpCBFIIIb and LpCBFIIIc. One major quantitative trait locus (QTL) was discovered in LpCBFIIIc for freezing tolerance, with the majority of the plants possessing the superior allele. Several minor loci were also discovered in this gene, but no significant loci were discovered in LpCBFIIIb.