Premise Different cytotypes of a species may differ in their morphology, phenology, physiology, and their tolerance of extreme environments. We studied the ecological niches of two subspecies of Saxifraga rosacea with different ploidy levels: the hexaploid Central European endemic subspecies sponhemica and the more widely distributed octoploid subspecies rosacea. Methods For both cytotypes, we recorded local environmental conditions and mean plant trait values in populations across their areas of distribution, analyzed their distributions by niche modeling, studied their performance at two transplant sites with contrasting conditions, and experimentally tested their cold resistance. Results Mean annual temperature was higher in hexaploid than in octoploid populations and experiments indicated that frost tolerance of the hexaploid is lower than that of the octoploid. Reproduction of octoploids from Central Europe was higher than that of hexaploids at a transplant site in subarctic Iceland, whereas the opposite was true in temperate Luxembourg, indicating adaptation of the octoploids to colder conditions. Temperature variables were also most important in niche models predicting the distribution of the two cytotypes. Genetic differences in survival among populations were larger for the octoploids than for the hexaploids in both field gardens, suggesting that greater genetic variability may contribute to the octoploid's larger distributional range. Conclusions Our results support the hypotheses that different cytotypes may have different niches leading to spatial segregation, and that higher ploidy levels can result in a broader ecological niche and greater tolerance of more extreme conditions.