Low- temperature (LT) tolerance is an economic trait in winter wheat that determines the plants’ ability to successfully cope with below freezing temperatures. To identify the genomic regions associated with LT tolerance in hexaploid wheat, an F2:3 population was produced from crossing between winter type and tolerant parent, Mirnovoskaja 808 (LT50 = -20oC), and spring type and susceptible parent, Pishtaz (LT50 = -7oC). The LT50, temperature at which 50% of the plants are killed by LT stress, was an LT tolerance index of F3 families. An F2:3 genotypes demonstrated a continuous distribution of trait values (LT50 = -3 to -23oC), in agreement with the distribution expected for a polygenic and quantitatively inherited trait. The results of molecular analyses for parent were assessed using 170 SSR primer pairs and 22 AFLP primer combinations. Seventy five polymorphic marker loci consisting of 20 SSR markers and 55 AFLP markers were selected for screening of F2 individuals. Linkage map was constructed using polymorphic markers. The markers were assigned to six linkage groups. Single marker analysis and interval mapping of phenotyped F2:3 individuals revealed a quantitative trait locus (QTL) on chromosome 5B. This locus explained 11.3% of the LT50 phenotypic variations.