Low temperature germination is an important agricultural trait. Improvement of crop tolerance to low temperature and identification of target genes are a major goal for crop breeders worldwide. By testing seed germination of over 300 Arabidopsis accessions, we identified a natural variation in germinability at low temperature in the dark among these accessions and compared the germination characteristics between Bay-0 and Shahdara. This natural variation was exploited to identify genetic loci responsible for cold-tolerant and dark germination. The molecular markers and a quantitative trait loci approach were used on recombinant inbred lines of a cross between Bay-0 and Shahdara. Six distinct quantitative trait loci were identified, three of which were major loci responsible for 61% of this natural variation. Validation of the three major loci using heterogeneous inbred families confirmed the feasibility of fine mapping and cloning the genes at the quantitative trait loci responsible for cold-tolerant and dark germination.