Prefrontostriatal and prefrontothalamic connections in rhesus monkeys have been shown to be organized in a topographic manner. These projections originate largely from infragranular layers V and VI. To examine whether the striatal and thalamic connections from the prefrontal cortex arise from separate neuronal populations or are collateralized, two different fluorescent retrograde tracers (diamidino yellow and fast blue) were injected into topographically similar regions of the head of the caudate nucleus and the mediodorsal nucleus in the same animal. The results show that although prefrontostriatal and prefrontothalamic projections arise from similar topographic regions, their laminar origins are distinctive. The connections to the head of the caudate nucleus originate mainly from layer Va, to a lesser extent from layer Vb, with a minor contribution from layers III and VI. In contrast, the projections to the mediodorsal nucleus emanate largely from layer VI, and also from layer Vb. Only occasional double-labeled neurons were observed, indicating that prefrontostriatal and prefrontothalamic connections originate from separate neuronal populations. The differential laminar distributions of neurons projecting to the head of the caudate nucleus and the mediodorsal nucleus suggest that these structures may receive independent types of information from the prefrontal cortex.