Plantago major and P. intermedia are two closely related inbreeding species. The isolation of polymorphic codominant microsatellite markers will provide valuable tools to investigate the reproductive isolation and the evolution of the two species. The isolation of microsatellite loci was achieved using a membrane enrichment method. Primers were designed to microsatellite flanking sequences and were analysed using fluorescent labels. Results indicated that nine out of the 10 loci amplified in both species, and that all the loci were polymorphic. The amplification of the loci was tested in a variety of Plantago species and was shown to be limited.