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This research developed a strategy for genetic improvement of the ectomycorhizal fungus Pisolithus microcarpus aiming at producing commercial innoculum for Eucalyptus seedlings. After mapping the spatial and temporal distribution of fruiting bodies in two experimental plots of a Eucalyptus understory, basidiospores were collected from six basidiomata and germinated to produce parental monokaryons isolates in vitro. After the identification of the sexual compatibility system of monokaryons, some compatibles parental isolates were crossed in vitro to produce hybrid dikaryotic progenies and to develop a model applicable to a diallell mating design. The diallell analyses were done for de trait of mycelia growth. The use of such model allowed the estimation of the values of General Combining Ability (GCA) of each parental monokaryons isolates and the values of Specific Combining Ability (SCA), for each one of the crossing system between parental monokaryons. These combining abilities showed the potential use of basiospores to produce commercial innoculum.