The colonization dynamics of the black truffle in an artificial field were assessed through analyses of microsatellite and RAPD markers. The truffle field was composed of three tree species and mycelial inoculum of three different origins, and was monitored for the first three years of truffle production. We found very low levels of genetic diversity. Isolation by distance was detected only at the between-tree level. This could be interpreted as local colonization around each tree facilitated by the presence of the tree root system. At the larger spatial scale of the European range, the absence of isolation by distance corroborates the hypothesis of an impact of glaciation on genetic variation, followed by rapid postglaciation demographic expansion. In addition, genetic variation of harvested truffles was explained by neither inoculation origin, nor tree species. Our study questions the real impact of man-made inoculation of tree root systems with fungal mycelia.