Molecular investigations of qualitative and quantitative changes in the genetic diversity of cultivated crops are useful for plant breeding and the management of crop genetic resources. A genotyping study, based on 28 genomic (g-SSR) and 13 expressed sequence tag-derived (e-SSR) microsatellite markers uniformly distributed across the barley genome, was conducted on samples of cultivated barley (Hordeum vulgare L.) collected at intervals of 40–50 years in three comparable geographical regions in Austria, Albania and India. The analysis indicated an absence of any significant differences either in the total number of alleles per locus or in g-SSR and e-SSR polymorphic information content (PIC) values from the Indian and Austrian materials. However, a slight reduction in genetic diversity was noted among the materials collected in Albania. The trend in numbers of collection mission-specific SSR alleles suggests significant allele flow over the time interval sampled. The g-SSRs yielded a higher mean number of alleles per locus and a superior PIC than the e-SSR markers. We conclude that a qualitative, rather than a quantitative shift in diversity has taken place over time, and that g-SSR markers detect more diversity than do e-SSR markers.