Thirty South American species of Cortinarius belonging to the subgenera Telamonia, Dermocybe, Myxacium, Phlegmacium, and Cystogenes were studied using an integrated approach that included morphological, anatomical, and ultrastructural data, and also molecular phylogenetic analysis of nuclear rDNA sequences. The micromorphology of the basidiomes was studied by light microscopy, and the principal structures were illustrated by line drawings. Basidiospore ornamentation was studied by scanning electron microscopy (SEM). Nuclear internal transcribed spacers (ITS, including the 5.8S gene) and the DNA coding for the D1/D2 domains of the large ribosomal subunit (LSU) were sequenced and analysed using a Bayesian Markov chain Monte Carlo method to estimate phylogenetic relationships between the studied Cortinarius species. Morphology and anatomy of the pileus surface and basidiome pigmentation appeared to be the most useful characters to delimit some natural groups, whereas microcharacters related to the structure of pileus context, hymenophoral and stipe trama were of little taxonomic value. Basidiospore morphology and cheilocystidia seem to be taxonomically relevant at the species level. The following five infrageneric groups were supported by the morphological, chemical and molecular data: (1) Telamonia characterized by wide hyaline hyphae of the veil and by small basidiomes; (2) Dermocybe spp. with an epicutis as the most external layer of the pileus, and skyrin and hypericin pigments; (3) Dermocybe spp. with a thin viscid layer on the pileus, and endocrocin and dermolutein pigments; (4) Phlegmacium spp. characterized by a long and radicating stipe; and (5) Phlegmacium spp. that overlap in some macrocharacters with Telamonia species. Our analyses suggest that classification concepts based mainly on macromorphological characters are likely to lead to artificial grouping, whereas certain microscopical and chemical characters seem to be useful in constructing a more natural classification system for Cortinarius.