Microsatellites (SSRs) are the most informative and popular class of molecular markers used for diverse purposes, particularly in plants: genetic diversity study, marker assisted selection, breeding, mapping, phylogenetics and phylogeography, systematics, etc. They have become a routine technique practically in each laboratory for studying molecular plant genetics. Despite their wide utilization, however, setup and optimization of various conditions involved in PCR amplification is a prerequisite for reliable inference of results. In this chapter, we describe optimization of SSR-PCR conditions and give ranges of concentrations for different parameters. The protocol provided here is inspired from bench work on the use of microsatellite to study diversity of Vitis vinifera germplasm.