Ceratocystis fimbriata sensu lato represents a complex of cryptic and commonly plant pathogenic species that are morphologically similar. Species in this complex have been described using morphological characteristics, intersterility tests and phylogenetics. Microsatellite markers have been useful to study the population structure and origin of some species in the complex. In this study we sequenced the genome of C. fimbriata. This provided an opportunity to mine the genome for microsatellites, to develop new microsatellite markers, and map previously developed markers onto the genome. Over 6000 microsatellites were identified in the genome and their abundance and distribution was determined. Ceratocystis fimbriata has a medium level of microsatellite density and slightly smaller genome when compared with other fungi for which similar microsatellite analyses have been performed. This is the first report of a microsatellite analysis conducted on a genome sequence of a fungal species in the order Microascales. Forty-seven microsatellite markers have been published for population genetic studies, of which 35 could be mapped onto the C. fimbriata genome sequence. We developed an additional ten microsatellite markers within putative genes to differentiate between species in the C. fimbriata s.l. complex. These markers were used to distinguish between 12 species in the complex.