Setting up a system for the genetic certification of chestnut cultivars requires the development of a reliable and inexpensive method of DNA typing. Microsatellite (= Simple Sequence Repeats) markers are considered particularly suitable for parentage studies and effective for the cultivar fingerprinting of tree species. The aim of the present work was to obtain a multiplex analysis of chestnut SSR loci by amplifying and electrophoretically separating several loci mixed together, using a semi-automated DNA sequencer and fluorochrome technology for the primer labeling. Initial results indicate the effectiveness of microsatellite analysis for DNA-typing of chestnut cultivars, and multiplex techniques can be used to reduce the cost of the work. Five SSR loci were characterized for size range and number of alleles in 96 individuals. In total 68 alleles were identified. These SSR loci are currently being used in multiplex analyses for the study of gene-flow between chestnut stands.