Ponderosa pine (Pinus ponderosa Dougl. ex P. & C. Laws.), an ectomycorrhiza (EM) dependent species, has been widely introduced in Patagonia, Argentina. This study used morphotyping, restriction analysis, and sequencing of EM root tips from ponderosa pine seedlings in two nurseries to assess the complete EM fungus (EMF) richness, to confirm doubtful identities of commonly reported morphotypes, and to evaluate the efficiency of morphotyping compared with molecular analysis. This interdisciplinary approach together with the fact that is the first study in which Patagonian nurseries EMF are genetically evaluated contributes to the general knowledge of this important group of fungi. Sequencing revealed the presence of 11 taxa. Basidiomycetes included Thelephoraceae (Tomentella sp.), Atheliaceae (Amphinema byssoides (Pers.) J. Erikss.), Hydnangiaceae (Laccaria sp.), Rhizopogonaceae (Rhizopogon roseolus (Corda) Th. Fr.), and Cortinariaceae (Hebeloma mesophaeum (Pers.) Quel.). Ascomycetes included Pezizaceae (Wilcoxina mikolae (Chin S. Yang & H.E. Wilcox) Chin S. Yang & Korf and Wilcoxina sp.) and Tuberaceae (Tuber sp.). Morphotyping proved to be useful for certain EMF species (R. roseolus, H. mesophaeum, A. byssoides, and to a lesser extent Tuber sp.) in which some morphological features are conspicuous and unique. Our detection of W. mikolae and Wilcoxina sp. are new records for ponderosa pine in Patagonia. All of the EM taxa identified are common to pine plantations and nurseries around the world, and no indigenous EM associated with native Nothofagus spp. were found.