Perennial ryegrass (Lolium perenne L.) has been used extensively for both forage and turf in temperate regions. To understand the genetic basis of winter hardiness and forage quality and molecular basis of cold acclimation of ryegrass, we mapped quantitative trait loci (QTL) that control winter hardiness and forage quality by using an interspecific ryegrass population and identified a C&barbelow;-repeat (CRT)/dehydration-responsive element (DRE) b&barbelow;inding f&barbelow;actors (CBF)-like gene in perennial ryegrass. Winter hardiness related traits including fall growth (FG), freezing tolerance (FT), and winter survival (WS) were measured over two years. A total of twenty-six QTLs were identified, of which seven were for FG, seven for FT, and thirteen for WS. The proportion of phenotypic variation explained by individual QTLs ranged from 10.4 to 22.1%. Coincident QTLs were detected on linkage groups (LGs) 4 and 5 for WS, on LG 5 for FT. To map QTLs that are associated with forage quality, neutral detergent fiber (NDF), acid detergent fiber (ADF), acid detergent lignin (ADL), and crude protein (CP) were determined by near-infrared reflectance spectroscopy (NIRS). Samples were harvested three times. Fiber components were all positively correlated with each other and negatively with CP. A total of 63 QTLs were detected. Coincident QTLs were detected on LGs 2, 6, and 7 for NDF, LGs 1, 2, and 7 for ADF, LGs 6 and 7 for ADL, and LG 2 for CP, respectively. The QTLs on LGs 2, 6, and 7 for fiber components were co-located on the same LG with several lignin biosynthetic genes from perennial ryegrass. A DREB1A/CBF3-like gene, designated LpCBF3, was isolated from perennial ryegrass by using RT-PCR and RACE. The LpCBF3 gene has an open reading frame of 233 amino acids and contains all the conserved domains known to exist in other CBF genes. Expression of the LpCBF3 is inducible by cold stress and overexpression of the LpCBF3 gene in Arabidopsis induced expression of the Arabidopsis DREB1A/CBF3 target COR genes and increased freezing tolerance of the transgenic plants without cold acclimation.