Technical Abstract: Ten polymorphic microsatellite loci for the oomycete obligate, biotrophic pathogen Peronospora tabacina of tobacco (Nicotiana tabacum) were identified from a small insert genomic library enriched for GT motifs. Eighty-five percent of the loci were composed of dinucleotide repeats, whereas only 4% and 11% were tri- and tetra-nucleotide repeats, respectively. About 82% of all the microsatellites were perfect and within the library, only about 7% of the loci were duplicated. Primers were designed for 63 loci; 10 loci were polymorphic, 19 were monomorphic, and 34 either failed to amplify or produced ambiguous/inconsistent results. The ten polymorphic loci were characterized using 44 isolates of P. tabacina collected from tobacco plants growing in Europe, the Near East, and North and South America. The number of alleles per locus was either 3 or 4 with a mean of 3.2 and the mean number of genotypes per locus was 3.6. Observed heterozygosity ranged from 0.32 to 0.95 and expected heterozygosity ranged from 0.44- 0.69 for these loci and all loci except PT054 did not conform to the Hardy-Weinberg distribution. Polymorphic information content (PIC) for the loci ranged from 0.35- 0.69 with a mean of 0.50. These microsatellite loci provide a set of markers sufficient to perform genetic diversity and population studies of P. tabacina, and possibly other species in the genus Peronospora and other genera of downy mildews.