Low temperature is one of the critical factors for determining agricultural production. Therefore, the main objective was to develop the durum wheat lines, which are tolerant to cold particularly in Central Anatolia. The recombinant inbred lines (RILs) (141) derived from F6 progeny of durum wheat, developed from Kunduru-1149 (female parent) and Cham-1 (male parent) cross, were characterized in terms of their cold tolerance both in the field and under controlled (in growth chamber) conditions. A 0–9 scale was used for the visual evaluation of cold damage under field conditions, and a quantitative measure of photochemical efficiency (Fv/Fm = variable fluorescence/maximum fluorescence) method was used for the evaluation of cold damage under controlled conditions. Seventeen RILs displayed the scores within the range of 1–2.5 in 0–9 scale evaluation, while 41 RILs showed the scores equal to or higher than 0.70 approaching to optimal (~0.83) in Fv/Fm evaluation. Some quality parameters tested and some gliadin fragments and random amplified polymorphic DNA loci showed meaningful correlations with the data of 0–9 scale and Fv/Fm ratios from lower to the medium level. The RILs considered as cold resistant; might have the potential to be used as parental lines to develop cold-resistant durum wheat varieties in the future durum wheat-breeding programmes.