The Autism Spectrum Quotient (AQ) has been widely used for measuring autistic traits however its factor structure has been primarily determined from nonclinic populations. This study aimed to establish an internally coherent and reliable factor structure for the AQ using a sample of 455 Australian adults of whom 141 had autism spectrum disorder (ASD) diagnoses. Principal component analysis revealed a 39-item questionnaire with five-factors: Sociability, Social Cognition, Interest in Patterns, Narrow Focus and Resistance to Change. The revised AQ-39 had sound goodness-of-fit indices, good-to-excellent internal consistency and test–retest reliability, and scores for ASD and non-ASD participants were significantly different. The AQ-39 may be useful in screening and for guiding the focus of therapy.