Background: Internalizing psychopathology, anxiety in particular, is assumed to contribute to the development of gender Identity disorder (GID). Until now, anxiety has only been reported in studies using parent-report questionnaires; physiological correlates of anxiety have not been studied. In this study we assessed anxiety and stress in children with GID by measuring their cortisol, heart rate (HR) and skin conductance levels (SCL) and asking them to repeat their moods and experience of control. Methods: By using an established psychological challenge, involving provocation and frustration, we investigated whether children with GID as compared to healthy controls react in a more anxious way under these experimental circumstances. We assessed anxiety levels in 25 children with GID and 25 matched controls by measuring cortisol, HR and skin conductance and examined whether a pattern of increased physiological activity corresponded with the feeling of being less in control and having more intense negative emotions. Results: The results showed that children with GID had more negative emotions and a tonically elevated SCL. There were no differences between the groups in cortisol and HR. Conclusion: This is the first study that shows that children with GID have a more anxious nature as compared to their normal counterparts.