The genetic composition and diversity of Plantago lanceolata L. populations were analysed using amplified fragment length polymorphism (AFLP) as well as simple sequence repeat (SSR) markers to test for differences in an old semi-natural grassland after five years of treatment with ambient or elevated ozone (O3) using a free-air fumigation system. Genetic diversity in populations exposed to elevated O3 was slightly higher than in populations sampled from control plots. This effect was significant for AFLP-based measures of diversity and for SSR markers based on observed heterozygosity. Also, a small but significant difference in genetic composition between O3 treatments was detected by analysis of molecular variance and redundancy analysis. The results show that micro-evolutionary processes could take place in response to long-term elevated O3 exposure in highly diverse populations of outbreeding plant species.