Legumes can be considered as pioneer plants during ecosystem development, as they form a symbiosis with different nitrogen fixing rhizobia species, which enable the plants to grow on soils with low available nitrogen content. In this study we compared the abundance and diversity of nitrogen fixing microbes based on the functional marker gene nifH, which codes for a subunit of the Fe-protein of the dinitrogenase reductase, in nodules of different size classes of Trifolium arvense (L.). Additionally, carbon and nitrogen contents of the bulk soil and plant material were measured. Plants were harvested from different sites, reflecting 2 (2a) and 5 (5a) yr of ecosystem development, of an opencast lignite mining area in the south of Cottbus, Lower Lusatia (Germany) where the artificial catchment "Chicken Creek" was constructed to study the development of terrestrial ecosystems. Plants from the 5a site revealed higher amounts of carbon and nitrogen, although nifH gene abundances in the nodules and carbon and nitrogen contents between the two soils did not differ significantly. Analysis of the nifH clone libraries showed a significant effect of the nodule size on the community composition of nitrogen fixing microbes. Medium sized nodules (2–5 mm) contained a uniform community composed of Rhizobium leguminosarum bv. trifolii, whereas the small nodules (< 2 mm) consisted of a diverse community including clones with non-Rhizobium nifH gene sequences. Regarding the impact of the soil age on the community composition a clear distinction between the small and the medium nodules can be made. While clone libraries from the medium nodules were pretty similar at both soil ages, soil age had a significant effect on the community compositions of the small nodules, where the proportion of R. leguminosarum bv. trifolii increased with soil age.