Laboulbeniales is one of the most peculiar oders of Ascomycota. These fungi are characterized by an ectoparasitic life-style on arthropods, determinate growth, lack of an asexual stage, high species richness, and intractability to culture. The order Laboulbeniales, sister to Pyxidiophorales, has only recently been assigned a separate class, the Laboulbeniomycetes, based on very fewribosomal DNA sequences. So far, DNA isolations and PCR ampli cations have proven dif cult. Here,we provide details of isolation techniques and the application of commercially available kits that enableef cient and reliable genetic analyses of these fungi. We provide 43 newly generated Laboulbenialesribosomal DNA sequences, among which are the rst published sequences for species in the generaGloeandromyces, Herpomyces, Laboulbenia, Monoicomyces, and Polyandromyces. DNA extractions were possible using from 1 to 30 thalli from hosts preserved in ethanol (70–100 %). In two cases, we successfully isolated DNA from thalli on dried insect collections. Laboulbeniales molecular systematics could be substantially enhanced through these improved methods by allowing more complete sampling of both taxa and gene regions.