The major outputs of the tectum appear fundamentally similar in all vertebrate species, suggesting that the major types of tectal output neurons have been evolutionarily conserved across a wide variety of vertebrate species. Nonetheless, tectal lamination patterns vary dramatically among vertebrates. To explore the basis of this variation in lamination at a neuronal level, the laminar distribution of the cells of origin of the major ascending and descending tectofugal pathways was determined in turtles using retrograde HRP labeling and the results compared to those from similar studies in diverse other species. Four major tectal outputs were studied: 1) the ipsilateral ascending projection to nucleus rotundus of thalamus; 2) the ipsilateral ascending projection to the dorsal and ventral lateral geniculate nuclei of the thalamus; 3) the crossed descending projection to paramedian regions of the pons and medulla; and 4) the ipsilateral descending projection to cell groups of the ventrolateral pons and medulla. The projection to nucleus rotundus was found to arise exclusively from multipolar neurons of the stratum griseum centrale, while the projection to the geniculate nuclei was found to arise from radial cells with long ascending dendrites in the stratum griseum periventriculare. The crossed descending projection to the paramedian hindbrain was found to arise almost exclusively from large multipolar neurons, the majority of which were located in the stratum griseum centrale and some of which were located in the stratum griseum periventriculare. This pathway was also found to give rise to a collateral projection that ascends in the ipsilateral hypothalamus. The ipsilateral descending projection was found to arise from multipolar neurons in the stratum griseum centrale and stratum griseum periventriculare, from radial and pyramidal neurons in the stratum griseum periventriculare, and from radial neurons in stratum griseum et fibrosum superficiale. These results show that the laminar distribution of the cells of origin of tectofugal pathways in turtles does not fit the simple notion that the midbrain roof is organized into superficial layers containing neurons with ascending projections and deep layers containing neurons with descending projections. Rather, individual layers in turtles appear to give rise to both ascending and descending projections, with there being evidence that each projection arises largely from a separate set of neurons. The pattern of distribution of the cells of origin of these various pathways is different in turtles than in either birds, mammals, frogs, salamanders or teleosts. Such results suggest that considerable interspecific variation occurs in the extent to which individual homologous neuronal populations migrate superficially during tectal development.