Brown knapweed (Centaurea jacea L.) has been suggested as a potential bioindicator for tropospheric ozone (O3), but little is known about the intra-specific variation in O3 sensitivity in this wild species. The aim of this study was to quantify the differences in O3 sensitivity among and within five populations, and to relate the differences to morphological, phenological, and genetic characteristics. These parameters were periodically recorded in two consecutive experiments on a total of 357 plants from five different European countries (Norway, Hungary, Switzerland, Italy, Slovenia). They were grown from seed in natural soil under ambient conditions at a site with seasonally elevated O3 concentrations (Cadenazzo, southern Switzerland). The populations differed significantly both in frequency and extent of O3 injury, as well as in phenological development. The observed degree of O3 injury was highest in the Slovenian and the Swiss populations, while only few Hungarian and Norwegian plants showed slight symptoms of injury. Plants were generally most sensitive to O3 when reaching the reproductive stage, and insensitive at the rosette stage. Amplified fragment length polymorphism analysis (AFLP) demonstrated genetic distinctiveness of the five C. jacea populations. All individuals of four of the five populations were correctly assigned to the respective populations based on principal component analysis. Cluster analysis quite accurately reflected the geographic origin of each population. Overall, the analysis revealed a high degree of intra-specific variability in O3 sensitivity in C. jacea, and underlined the important influence of the climate-dependent population-specific plant development on O3 sensitivity. These observations may constrain the development of a standardized biomonitoring system.