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Existing measures of communication challenges in autism are based on diagnostic criteria and research/clinical observations of autistic people, rather than what autistic people themselves identify as difficulties. In this study, the Conversation Questionnaire (CQ) was developed based on community engagement with autistic people to identify what they find challenging about conversation. This new tool was then administered online to autistic, dyslexic and neurotypical people ( N  = 312) in a validation phase of the study. Item-response theory modelling indicated that a two-dimensional structure accounted for response patterns. These dimensions reflected difficulties knowing what to say (15 items) and engaging in behaviours possibly disruptive to neurotypical conversation (21 items). The dimensions showed good internal consistency and convergent and discriminant validity, and could distinguish between autistic and neurotypical people ( d  = 1.59 and d  = 2.07 respectively). The CQ might help contribute to diagnostic assessment for autism in adults as part of a holistic assessment. The questionnaire might also be useful with other neurodiverse groups, and provide a tool for clinicians and researchers to identify individuals’ strengths and difficulties in conversation (e.g., as part of interventions in speech and language therapy).