Background: Comorbidity of anxiety and depression predicts impaired treatment outcomes, poor quality of life and increased suicide risk. No study has reported on a combined measure of anxiety-depression in boys with an Autism Spectrum Disorder. Aims: To explore the prevalence, underlying factor structure and relationships between anxiety-depression, physiological stress and symptoms of Autism Spectrum Disorder (ASD). Methods: 150 boys (aged 6-18 years; IQ M= 94.9, range. = 73-132) with an ASD plus their parents (135 mothers, 15 fathers) completed scales about the boys' anxiety and depression, and the boys provided samples of their saliva in the morning and afternoon. Parents also completed the ASD Behaviour Checklist about the boys' ASD symptoms. Results: The two sources of ratings were not significantly different for prevalence of anxiety-depression but the factor structures varied between the parents' and boys' responses, with a four-factor solution for the boys' ratings and a three-factor solution for the parents' ratings. There were also differences in the correlations between cortisol and anxiety-depression and between ASD symptoms and anxiety depression across the boys' and parents' data. Conclusions: Assessment of anxiety and depression comorbidity from parents and from children with an ASD themselves could provide a valuable adjunct datum when diagnosing ASD.