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The inclusive education framework and the increase in autism diagnoses have led to an overwhelming challenge for pre-service teachers who need to be qualified to teach all children. To test the quality of their training, the main purpose of this study was to compare 866 pre-service teachers’ knowledge, misconceptions, and gaps about autism in their first and final year at university, using the Autism Knowledge Questionnaire. The results show that fourth-year students obtained higher levels of knowledge and fewer gaps than the first-year students, although they also had more misconceptions. Special education specialists obtained significantly more knowledge and fewer misconceptions than the general education pre-service teachers. Specific training and experience had a significant influence on the knowledge and gaps, but it had no influence on the number of misconceptions. These results suggest that university preparation in autism spectrum disorder (ASD) might not adequately train all future teachers.