Primula vulgaris is a perennial herb from moist native forest vegetation. In western European lowlands, it has become rare, still declining and occurring in forest remnants embedded within an agricultural matrix. In Belgium and The Netherlands, it received special attention for conservation. We developed 12 novel polymorphic microsatellite loci and two multiplex PCR including 15 loci. For seven European populations, they displayed 7–28 alleles. Observed and expected heterozygosities at population level ranged from 0.28–0.61 and 0.24–0.70, respectively. Genetic differentiation was high between western, central and eastern European populations. The variability recorded will allow investigating genetic diversity and structure patterns at local and continental scales, but also quantifying gene dispersal at a fine scale to estimate effects of habitat fragmentation on connectivity.