To support conservation policies for old Dutch grasslands that are still in agricultural use, morphological variation and AFLP-based (amplified fragment length polymorphism-based) genetic diversity was studied in perennial ryegrass and white clover populations and compared with the diversity in reference varieties. In addition, AFLP variation was also studied in grasslands located in nature reserves. From principal component analysis (PCA), it appeared that date of ear emergence in perennial ryegrass and characters related to plant vigour in white clover were the main morphological characters separating the reference varieties from the old Dutch grassland populations, and some of the grassland populations from each other. In both species, intrapopulation variation was lower for the reference varieties. Lower heterogeneity within the reference varieties was also found in the AFLP analysis. All common AFLP's observed in old Dutch grasslands could also be found in the reference varieties and nature reserves. Only a small number of low-frequency alleles found in old Dutch grasslands were absent from the other two groups. However, band frequencies of markers could vary considerably between populations, which may have been caused by selection. Analysis of the AFLP data by PCA distinguished the majority of reference varieties from the old Dutch grasslands, and showed genetic differentiation only between some grasslands. Comparison of old Dutch grasslands with grasslands in nature reserves indicated that basically the same range of genetic variation is covered by the two groups. Our study indicates that the Netherlands harbour a more or less continuous population for major parts of the diversity of perennial ryegrass and white clover. It was concluded that no specific conservation measures are presently needed to maintain genetic diversity of perennial ryegrass and white clover occurring in old Dutch grasslands.