Abstract The New World marsupial family Didelphidae is one of the oldest among mammals and is usually regarded as a morphologically conservative group. We analyzed cranial shape variation among six species of the six largest living genera of the family using two-dimensional landmark data. We captured and digitized video images of the skull and mandible for the following species: Caluromys philander (n = 65), Chironectes minimus (n = 30), Didelphis aurita (n = 70), Lutreolina crassicaudata (n = 37), Metachirus nudicaudatus (n = 77) and Philander frenata (n = 62). Fourteen landmarks were defined for the lateral, 25 for the ventral, 23 for the dorsal views of the skull, and nine on the mandibular lateral view. Sex, species, and interaction effects were analyzed with a two-way MANOVA on the matrices of coordinates aligned by general least squares. All four views had significant interactions. Canonical Variates Analysis was performed on sexes and species, and shape was regressed on the canonical variate scores for each species. Caluromys philander was clearly the most distinct species, with paedomorphic features that can be related to its arboreal habits. A conspicuous shortening of the rostrum distinguishes the highly carnivorous Lutreolina crassicaudata. Didelphis aurita and Philander frenata overlapped somewhat, reflecting shape similarities associated with their phylogenetic affinities, while the few differences observed are probably allometric consequences of size differences. Philander frenata and Chironectes minimus showed similar cranial shapes, while Metachirus nudicaudatus was distinctive with a broad and elongated rostrum. In spite of an overall similar shape, the geometric morphometric approach revealed several marked differences among species that can be related to their phylogenetic origin and their adaptive zone.