AbstractAims – Centaurea cyanus L. is a patrimonial species emblematic of the flora associated with cereal crops, but it is currently declining in Western Europe because of agricultural intensification. The decrease in both the area and connectivity of populations could affect the genetic structure and variability of the populations, and in turn their potential to survive. Methods – We studied the genetic variability of ten large and five small C. cyanus populations from two contrasted French regions using seven nuclear isozyme markers, and of twelve Western European populations using eighteen chloroplast markers. Results – The analysis of leaf isozymes revealed that all populations, except one, had a deficit of heterozygous individuals (average Fis = 0.81), which could confirm the presence of a proportion of self-compatible plants. The level of variability was high. Genetic diversity was not lower in small than in large populations, and there was no reduced genetic diversity in the region where C. cyanus populations are rare. There was low but significant differentiation among populations (Fst = 0.068), especially between small and large populations, but not between regions. In contrast, no polymorphism was found among the 371 chloroplast DNA sequences analysed (i.e. around 8–10% of the chloroplast genome). Conclusions – The observed pattern of variability could reflect the first effects of the rarefaction and fragmentation of previously abundant, widely distributed and poorly structured C. cyanus populations, on the reproduction regime and onset of genetic heterogeneity among populations.