Monilinia species causing disease on stone and pome fruits have overlapping and similar host-ranges, disease symptoms and morphology. It can be difficult to reliably differentiate among native and exotic (regulated) species. HTS sequencing (Third Generation Sequencing; TGS) uses single molecule templates that have a higher error rate than alternatives, but that provide longer read lengths. TGS long reads can be used to create draft genomes with better assembly and increased potential for the discovery of structural variations and diagnostic markers. To better characterize a Monilinia genome, we used TGS to sequence an isolate of M. fructigena (Mfg5-SP-A). This is an exotic pathogen under regulatory control for the United States. We generated a PacBio and Mi-Seq hybrid genome assembly, then annotated the genome to identify unique targets between Monilinia species. We used comparative genomics with a custom Monilinia Genomics Database (MGDb) to predict more than 9600 genes in M. fructigena. We identified approximately 350 genes that represent potential diagnostics targets to distinguish among the especially closely related M. fructigena and M. polystroma. A more exhaustive comparison suggests that the number of truly discriminating targets may be considerably lower than those ~350. Our analysis for unique and common predicted genes shows all possible logical relations between 4 Monilinia species (two native and two exotic) that could be used to identify DNA markers and functional genes with potential to be used for diagnostic purposes.