Light and temperature are key external factors in the control of Arabidopsis thaliana seed germination and dormancy mechanisms. Perception and response to these stimuli have to ensure that seedling emergence and growth occur at the most advantageous time for correct establishment. Analysis of over 300 Arabidopsis accessions identified 14, from 12 different geographical locations, that were able to germinate to greater than 20% at 6 degrees C in the dark. This natural variation was exploited to identify genetic loci responsible for cold-tolerant, dark germination. A quantitative trait loci approach was used on recombinant inbred line progeny of a cross between Bay-0 and Shahdara. Six distinct quantitative trait loci were identified, three of which were major loci, each responsible for 17-25% of the phenotypic variability in this trait. Parental phenotypes indicated that the majority of the cold-tolerant, dark-germination characteristics are related to light responses. 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, dark germination.