Papaver rhoeas, an annual plant species in the Papaveraceae family, is part of the biodiversity of agricultural ecosystems and also a noxious agronomic weed. We developed microsatellite markers to study the genetic diversity of P. rhoeas, using an enriched microsatellite library coupled with 454 next-generation sequencing. A total of 13,825 sequences were obtained that yielded 1795 microsatellite loci. After discarding loci with less than six repeats of the microsatellite motif, automated primer design was successful for 598 loci. We tested 74 of these loci for amplification with a total of 97 primer pairs. Thirty loci passed our tests and were subsequently tested for polymorphism using 384 P. rhoeas plants originating from 12 populations from France. Of the 30 loci, 11 showed reliable polymorphism not affected by the presence of null alleles. The number of alleles and the expected heterozygosity ranged from 3 to 7.4 and from 0.27 to 0.73, respectively. A low but significant genetic differentiation among populations was observed (F-ST = 0.04; p < 0.001). The 11 validated polymorphic microsatellite markers developed in this work will be useful in studies of genetic diversity and population structure of P. rhoeas, assisting in designing management strategies for the control or the conservation of this species.