We investigated the structural and molecular organization of the extracellular matrix in Thylamys elegans, a marsupial representative of the mammalian order Didelphimorphia. Perineuronal nets (PNs) associated with distinct types of neurons were visualized by detection of chondroitin sulfate proteoglycans and hyaluronan, and by labeling with Wisteria floribunda agglutinin (WFA), a marker for PNs in the mammalian brain. In the neocortex of Thylamys, these methods revealed PNs on pyramidal cells. In contrast, parvalbumin-immunoreactive interneurons in the neocortex and hippocampal formation (displaying robust, WFA-labeled PNs in placental mammals) were ensheathed only with a delicate rim of hyaluronan and proteoglycans not detectable with WFA. The absence of WFA staining was characteristic also of some subcortical regions which contained PNs intensely labeled for chondroitin sulfate proteoglycan and hyaluronan. However, corresponding to placental mammals, numerous subcortical nuclei showed clearly WFA-stained PNs. Similar as in placental mammals, cholinergic basal forebrain neurons and tyrosine hydroxylase-immunoreactive neurons of the substantia nigra and locus coeruleus were devoid of PNs. Together with our earlier study on Monodelphis, the present results reveal that South American opossums show either a particular "marsupial" or "Didelphid" type of extracellular matrix chemoarchitecture, supporting the view that these components may vary phylogenetically as integral parts of neuronal physiology at the systems and single cell level.