The objective of this study was to investigate the effect of one cycle of seed regeneration on the conservation of genetic diversity in five Italian ryegrass landraces (Lolium multiflorum Lam.). Regeneration took place outdoors, in a sheltered site surrounded by tall Galician wheat, 20 m from the nearest source of alien pollen. A balanced mixture of seed (the same weight of seed per plant) was made from 90–100 plants harvested within each population. The conservation of allele frequencies was assessed by starch gel electrophoresis. Five enzyme systems from 78–153 plants per population were examined on slices of a single histidine–citrate starch gel. Each regenerated population differed from its original landrace in at least one of the five loci. The mean heterozygosity per locus was 0.45 for original and regenerated populations, and the mean number of alleles per locus was 3.7 and 3.6 for original and regenerated populations, respectively. There was no loss of common alleles (frequency >0.05) in the five regenerated populations compared with the original populations. Only three rare alleles (frequency < 0.05) were lost (e.g. alleles phosphoglucose isomerase (PGI)-2a, PGI-2c* and shikimate dehydrogenase (SDH)-1d in Padrón, Pravia and Luarca, respectively). No regeneration effect (P>0.05) was observed in the six agromorphological characters. However, a significant landrace effect was observed (P < 0.05) in the five agromorphological traits and the regenerated landraces deviated from the original landraces in 20% of direct comparisons. The results suggest that the method of regeneration used was not very suitable for maintaining the genetic integrity of the original landraces.