Plant roots are inhabited by an enormous variety of microorganisms, including fungi, which can control the growth as well as regulate the health of the host plants. The mycobiome composition of the roots of wheat plants, especially spelt, under drought stress has been rarely investigated. Therefore, the aim of the present study was to examine the composition of fungal communities in the root endosphere and rhizosphere of three Triticum aestivum ssp. spelta L. cultivars and one Triticum aestivum ssp. vulgare L. cultivar, grown under drought and controlled conditions in different soil preparations. Culture-dependent fungal community profiling was performed to examine the impact of rhizocompartments (endosphere, rhizosphere), host genotype, watering status and different soil preparation on roots mycobiome structure. A total of 117 fungal strains, belonging to 22 genera, were found to colonize the internal and external parts of roots in T. aestivum ssp. spelta L. and T. aestivum ssp. vulgare L. cultivars. The results showed that the part of root and soil preparation type significantly determined the mycobiome composition of wheat roots.