Forest trees rely on ectomycorrhizal (ECM) fungi for acquiring scarce resources such as water and nutrients. However, the molecular mechanisms governing ECM traits remain inadequately understood, particularly the role of intraspecific fungal variation in root-tip colonisation and trophic interactions. This study examined six ECM traits using Pisolithus microcarpus, an ECM fungus capable of forming ECM rootlets in poplar. A collection of 40 sibling monokaryons and their parental dikaryon was analysed through genome and transcriptome sequencing to examine quantitative trait loci, gene expression and mating-type loci. These findings revealed a pronounced phenotypic continuum in poplar root colonisation by sibling monokaryons, ranging from incompatible to fully compatible strains. Genetic recombination among monokaryons was demonstrated, and genomic regions potentially involved in ECM fungal traits were identified. Transcriptomic analysis revealed greater differentiation in transcriptomic profiles between fungal strains than between fungal tissues, and uncovered tissue-specific functional responses for ECM and free-living mycelia. Poplar exhibited distinct transcriptomic responses when interacting with different sibling monokaryons and the parental dikaryon. Allele sorting at 11 mating-type loci confirmed the species' heterothallic tetrapolar system. This study advances understanding of the genetic and transcriptomic mechanisms underlying ECM symbioses, highlighting intraspecific fungal diversity's role in forest ecosystem functioning.