Germplasm conserved as seeds in genebanks requires regular regeneration. In this process, selection and genetic drift may cause loss of genetic diversity from accessions. In the case of selfing crops, separation of distinct lines into different accessions may be an efficient strategy to avoid these negative effects. In order to evaluate the applicability of this method for collection management, knowledge about the level of intra-accession genetic diversity is required. By means of AFLP analysis intra-accession variation was investigated in two cultivars, two landraces and two wild populations of ex situ conserved barley germplasm. In the total sample of 216 individuals analysed (36 per accession), 22 genotypes were observed based on 104 polymorphic loci. The number of genotypes detected ranged from 1 to 3 per accession, except for a Nepalese landrace that revealed 12 genotypes. An UPGMA cluster analysis grouped the genotypes unambiguously into the accession they belonged to and genotypes within accessions were generally found to be closely related. In order to determine the repeatability of the results obtained, 11 individuals belonging to 4 genotypes from the Nepalese landrace were scored for a second set of AFLP markers. Matrices of genetic distances calculated for the two AFLP datasets were found to be highly correlated (r = 0.9346, P < 0.001). Separation of genotypes into different accessions was considered a relevant option only for the Nepalese landrace. Analysis of molecular variance indicated that this accession could be well divided into 8 distinct lines. Further implications of the results for genebank practices are discussed.