Root alkaloids remain highly unexplored in ectomycorrhizae development studies. By employing ultrahigh mass resolution mass spectrometry imaging techniques, we showed substantial relocation and transformation of piperidine alkaloids in pine root tips in response to Suillus mycorrhization. We imaged, in the time frame of ectomycorrhizae formation, a completely different alkaloid profile in Pinus strobus, where basidiospores of Suillus spraguei induce morphogenesis of symbiotic tissues, than in Pinus taeda, where such interaction fails to induce morphogenesis. On the basis of spatial colocalization studies, we proposed some alternative routes for biosynthesis of these alkaloids that supplement existing literature data.