Large numbers of crop plant accessions from all over the world have been amassed in gene banks to secure a gene pool for future breeding programmes. Maintenance of accessions held as seed samples in cold stores involves frequent rejuvenation cycles to ensure the viability of seeds. The practice of rejuvenation by multiplication of a sample of each accession in small field plots has the potential to create population bottlenecks, leading to loss of genetic diversity and changes in gene frequencies every rejuvenation cycle. In order to determine whether these undesirable effects occur, genetic diversity levels were assessed for morphological and isozyme markers within gene bank accessions of two barley landraces from Syria that had been stored for 10, 40 and 72 years. These were compared with genetic diversity levels for the same markers in barley landraces collected recently at locations in Syria where they are still under cultivation. Average gene diversity (H), alleles per locus (A) and percentage polymorphic loci (P (0.01)) all showed very significant declines with length of time in storage, and genetic differentiation FST among accessions increased over time. If the observed differences in genetic diversity are caused by genetic drift in gene bank accessions rejuvenated every 5.3 years, it was estimated that the effective population size Ne of rejuvenation populations over their period in storage was only 4.7. Implications for gene bank management are discussed.