Rat pups that are repeatedly handled and separated from their dam exhibit altered adult behavioral, endocrine, and autonomic responses to stress, but the extent to which early handling and/or maternal separation (H/S) alters the development of circuits that underlie these responses is unknown. The present study tested the hypothesis that early H/S alters the postnatal assembly of synapses within preautonomic emotional motor circuits. Circuit development was traced by synapse-dependent retrograde transneuronal transport of pseudorabies virus (PRV) from the stomach wall. Control and H/S rats were analyzed between postnatal day 6 (P6) and P10, a period of rapid synaptic assembly among preautonomic circuit components. Pups in H/S groups were removed from their dam daily for either 15 min or 3 h beginning on P1, and were injected with virus on P8 and perfused on P10. Quantitative analyses of primary and transsynaptic PRV immunolabeling confirmed an age-dependent assembly of hypothalamic, limbic, and cortical inputs to autonomic nuclei. Circuit assembly was significantly altered in H/S pups, in which fewer neurons in the central amygdala, the bed nucleus of the stria terminalis, and visceral cortices were infected compared with age-matched controls. In contrast, H/S did not alter the assembly of paraventricular hypothalamic inputs to gastric autonomic neurons. H/S-related reductions in limbic and cortical transneuronal infection were similar in pups exposed daily to 15 min or 3 h maternal separation. These findings support the view that environmental events during early postnatal life can influence the formation of neural circuits that provide limbic and cortical control over autonomic emotional motor output.