The cornflower, Cyanus segetum Hill, 1762 (formerly Centaurea cyanus L.) is an iconic arable plant that was historically widespread in Western Europe, but has shown a marked demographic decline since the mid-20th century. The species is distinctively attractive due to its large blue-coloured flowers and is an important source of nectar for pollinating insects. For these reasons, populations from horticultural or cultivated origins are frequently sown within flower fallows and flower strips. However, cultivated populations may be of non-local origin and have a reduced and/or divergent genetic variation. Therefore, replacement of local populations by cultivated ones or gene flow from cultivated populations to wild populations may represent a threat for the in situ conservation of wild cornflower. In this study, we described and compared the genetic diversity and genetic structure of wild populations sampled throughout France with that of plants sampled in flower fallows and from commercial seed lots. Plants originating from more than fifty wild populations, seven flower fallows and twelves commercial seed lots were genotyped with seven microsatellite markers. A large genetic diversity and relatively low genetic differentiation (Fst = 0.128) was revealed among wild populations, indicating a rather low genetic impact of demographic regression and population fragmentation on cornflower. Most horticultural populations had a different genetic makeup than wild populations. Flower fallows were genetically intermediate, suggesting that genetic mixing between wild and horticultural sources takes place within them. As this may negatively impact the local adaptation of wild populations, we advocate that cornflower seeds of wild, local origin are used in seed mixtures for flower fallows and flower strips.