Background: Separation anxiety disorder (SAD) is one of the most common anxiety disorders in childhood and is predictive of adult anxiety disorders, especially panic disorder. However, the disorder has seldom been studied and the attempt to distinguish SAD from other anxiety disorders with regard to psychophysiology has not been made. We expected exaggerated anxiety as well as sympathetic and respiratory reactivity in SAD during separation from the mother. Method: Participants were 49 children with a principal diagnosis of SAD, 21 clinical controls (CC) with a principal diagnosis of anxiety disorder other than SAD, and 39 healthy controls (HC) not meeting criteria for any current diagnosis. Analyses of covariance controlling for age were used to assess sympathetic and parasympathetic activation (preejection period and respiratory sinus arrhythmia) as well as cardiovascular (heart rate, mean arterial pressure, total peripheral resistance), respiratory (total breath time, minute ventilation, tidal volume, end-tidal CO[subscript 2], respiratory variability), electrodermal, and self-report (anxiety, cognitions, symptoms) variables during baseline, 4-min separation from, and reunion with the mother. Results: Children with a diagnosis of SAD were characterized by elevated self-reported anxiety responses to separation and increased sympathetic reactivity compared with CC and HC groups. The SAD group also displayed greater vagal withdrawal and higher reactivity in multiple cardiovascular, respiratory, and electrodermal measures compared with the HC group, while corresponding responses were less in the CC group and not significantly different from the other groups. Conclusions: Separation from the mother elicits greater autonomic, respiratory, and experiential responses in children with SAD. Our findings based on brief experimental separation demonstrate differential subjective and physiological manifestations of specific anxiety diagnoses, thus supporting the validity of the diagnostic category of SAD.