The genetic variation of Duddingtonia flagrans, which has become a promising biocontrol agent of animal parasitic nematodes, was investigated in a worldwide collection of 22 isolates. We analysed the sequence variation in four nuclear genes, tubA (β-tubulin), CMD1 (calmodulin), EF1α (translation elongation factor 1α), and PII (extracellular serine protease). 1428 aligned base pairs (bp) were analysed from the four genes, including 709 bp of introns. In addition, the variations in three anonymous genomic regions comprising 1155 bp were examined. Three single nucleotide polymorphisms (SNPs) were detected in the seven loci, none of them in the protein encoding genes. The genetic variation was significantly higher in the nematode-trapping fungus Arthrobotrys oligospora, the closest evolutionary relative to D. flagrans. Analysis of 12 isolates of A. oligospora revealed 30 SNPs in tubA, CMD1, EF1α and PII. The genetic variation in the isolates of D. flagrans was further examined using AFLP analysis. Five primer combinations were used to detect 159 bands, of which 94 (59.1%) were polymorphic. A neighbour-joining tree based on the AFLP data showed no clear association between genotype and geographical origin. Furthermore, the AFLP data suggest that D. flagrans is mainly clonal and no recombination could be detected, not even within the same country. The low genetic variation in D. flagrans suggests that this fungus has recently diverged from a single progenitor. Based on estimations of mutation rates, it was calculated that this most recent common ancestor lived about 16000–23000 years ago.