Tuber aestivum, also known as the summer or Burgundy truffle, is an ectomycorrhizal Ascomycete associated with numerous trees and shrubs. Its life cycle occurs in the soil, and thus soil parameters such as temperature and water availability could influence it. T. aestivum cultivation has started in several countries, but ecological and agronomic requirements for the establishment and management of orchards are largely unknown. The aims of this work were: 1) to design a specific qPCR protocol using genomic data to trace and quantify T. aestivum DNA in the soil; and 2) to assess the monthly soil DNA dynamic according to soil parameters (i.e. soil hydric potential and temperature) in this orchard. The study was conducted in a highly productive T. aestivum orchard (hazels, oaks, pines, lime and hornbeam). The production started five years after the plantation and then increased exponentially to reach a maximum of 320 kg/ha in 2017. The soil hydric potential and temperature partially explained the monthly T. aestivum soil DNA variability. The data presented here offer new insights into T. aestivum ecology and cultivation.