Abstract: The genus Metarhizium includes insect pathogenic fungal species, which are currentlyused as biological control agents. The development of effective biological control strategiesrequires detailed knowledge of their biology and the environmental factors affecting theirdistribution and abundance. The goal of this study was to monitor Metarhizium spp. includingM. anisopliae, M. pinghaense, M. majus, M. brunneum, M. robertsii, and M. guizhouense in soilusing a cultivation-independent PCR-based detection and quantification tool. Soil was collectedfrom different habitat types including semi-natural habitats, which may provide a refuge forMetarhizium spp. Abundance of Metarhizium spp. was significantly higher in semi-naturalhabitats, i.e., improved field margins and permanent grassland compared to cereal fields.Furthermore, uneven distribution of Metarhizium spp. was observed. Assessment of correlationsbetween PCR and cultivation-dependent based data demonstrated the reliability and robustness ofthe PCR-based tool. The findings illustrated the value of fast and reliable detection andquantification tools for analyzing large numbers of soil samples and for further investigations offactors causing the uneven distribution of Metarhizium spp.