Forty-one microsatellite markers were developed for the poplar rust fungus Melampsora larici-populina. The genome sequence of M. larici-populina strain 98AG31 was screened in silico for microsatellite loci using Magellan software. Thirty-nine loci, evaluated on a sexual population of 35 M. larici-populina isolates sampled on larch trees in the French Alps, were identified to be highly polymorphic. These markers revealed two to 34 alleles, with an average of 7.23 alleles per locus. The estimated heterozygosity ranged from 0.029 to 0.958 across the 41 microsatellite loci. These markers will be highly useful for population genetics studies of M. larici-populina.