Main conclusion Higher acclimated freezing tolerance improved winter survival, but reduced reproductive fitness of Arabidopsis thaliana accessions under field and controlled conditions. Abstract Low temperature is one of the most important abiotic factors influencing plant fitness and geographical distribution. In addition, cold stress is known to influence crop yield and is therefore of great economic importance. Increased freezing tolerance can be acquired by the process of cold acclimation, but this may be associated with a fitness cost. To assess the influence of cold stress on the fitness of plants, long-term field trials over 5 years were performed with six natural accessions of Arabidopsis thaliana ranging from very tolerant to very sensitive to freezing. Fitness parameters, as seed yield and 1000 seed mass, were measured and correlation analyses with temperature and freezing tolerance data performed. The results were compared with fitness parameters from controlled chamber experiments over 3 years with application of cold priming and triggering conditions. Winter survival and seed yield per plant were positively correlated with temperature in field experiments. In addition, winter survival and 1000 seed mass were correlated with the cold-acclimated freezing tolerance of the selected Arabidopsis accessions. The results provide strong evidence for a trade-off between higher freezing tolerance and reproductive fitness in A. thaliana, which might have ecological impacts in the context of global warming.