Autism spectrum disorder shares many symptoms with other mental health disorders, and comorbid disorders such as mood and anxiety disorders are common, making the diagnostic process challenging. We aimed to explore the diagnostic accuracy of two standard autism spectrum disorder diagnostic instruments and to identify those behavioral items that best differentiate between autism spectrum disorder and mood and anxiety disorder in a naturalistic sample of patients utilizing autism spectrum disorder specialist services. The study included data of 847 participants (5-65 years of age, n = 586 with autism spectrum disorder, n = 261 with mood and anxiety disorder) all evaluated with the Autism Diagnostic Observation Schedule in the context of the diagnostic process. Data of the Autism Diagnostic Interview--Revised were available for 428 participants (5-51 years of age, n = 367 with autism spectrum disorder, n = 61 with mood and anxiety disorder). By means of binominal logistic regressions and an ensemble feature selection, we identified a subset of items that best differentiated between autism spectrum disorder and mood and anxiety disorder. Overall, the results indicate that a combination of communicational deficits and unusual and/or inappropriate social overtures differentiates autism spectrum disorder and mood and anxiety disorder. Aspects of social cognition are also relevant. Limitations of the current study and implications for research and practice are discussed.