The Périgord black truffle (Tuber melanosporum Vittad.), considered a gastronomic delicacy worldwide, is an ectomycorrhizal filamentous fungus that is ecologically important in Mediterranean French, Italian, and Spanish woodlands. In this study, we developed a novel resource of single nucleotide polymorphisms (SNPs) for T. melanosporum using Illumina high-throughput re-sequencing. The genome from six T. melanosporum geographic accessions was sequenced to a depth of ~20×. These geographic accessions were selected from different populations within the northern and southern regions of the geographical species distribution. Approximately 80% of the reads for each of the six re-sequenced geographic accessions mapped against the reference T. melanosporum genome assembly, estimating the core genome size of this organism to be ~110 Mbp. A total of 442,326 SNPs corresponding to 3,540 SNPs/Mbps were identified as being included in all seven genomes. The SNPs occurred more frequently in repeated sequences (85%), although 4,501 SNPs were also identified in the coding regions of 2,587 genes. Using the ratio of non-synonymous mutations per non-synonymous site (pN) to synonymous mutations per synonymous site (pS) and Tajima’s D index scanning the whole genome, we were able to identify genomic regions and genes potentially subjected to positive or purifying selection. The SNPs identified represent a valuable resource for future population genetics and genomics studies.