This essay reexamines molecular evidence supporting the existence of the ‘preisthmus’, a caudal midbrain domain present in vertebrates (studied here in the mouse). It is thought to derive from the embryonic m2 mesomere and appears intercalated between the isthmus (caudally) and the inferior colliculus (rostrally). Among a substantial list of gene expression mappings examined from the Allen Developing and Adult Brain Atlases, a number of quite consistent selective positive markers, plus some neatly negative markers, were followed across embryonic stages E11.5, E13.5, E15.5, E18.5, and several postnatal stages up to the adult brain. Both alar and basal subdomains of this transverse territory were explored and illustrated. It is argued that the peculiar molecular and structural profile of the preisthmus is due to its position as rostrally adjacent to the isthmic organizer, where high levels of both FGF8 and WNT1 morphogens must exist at early embryonic stages. Isthmic patterning of the midbrain is discussed in this context. Studies of the effects of the isthmic morphogens usually do not attend to the largely unknown preisthmic complex. The adult alar derivatives of the preisthmus were confirmed to comprise a specific preisthmic sector of the periaqueductal gray, an intermediate stratum represented by the classic cuneiform nucleus, and a superficial stratum containing the subbrachial nucleus. The basal derivatives, occupying a narrow retrorubral domain intercalated between the oculomotor and trochlear motor nuclei, include dopaminergic and serotonergic neurons, as well as a variety of peptidergic neuron types.