This study was conducted to identify physiological traits associated with cold tolerance in sunflower and to identify the genomic regions involved in their variation. A population of 98 recombinant inbred lines (RILs) and their two parents were sown in the field as usual sowing date (control) and one or two months earlier (long-term low temperature treatments). A trait commonly used to underlying cold tolerance related to the degree of membrane damage, as well as traits associated with growth capacity (chlorophyll content, potential photochemical efficiency of photosystem II and plant dry weight) and finally those reflecting acclimation mechanism to stress conditions (osmotic potential at full turgor, and specific leaf area) have been investigated at early development stages. Significant differences were observed among the three sowing dates for all traits. Chlorophyll content and specific leaf area are genetically associated with cold tolerance. Genetic gains were observed for chlorophyll content and osmotic potential traits in some of early sowing dates, which suggest that they could be used for cold tolerance in breeding programs. QTL analyses show that several putative genomic regions are involved in the variation of the physiological traits studied under low temperature. Major QTLs for cold tolerance associated with SSR markers such as ORS331_2 for the cell membrane stability should be checked in several environments to see if they can be used in marker-assisted selection programs.