Abstract Background Autism is a neurodevelopmental disorder that is first manifested during early childhood. Postmortem experiments have identified significantly elevated expression of metabotropic glutamate receptor 5 (mGluR5) in cerebellar vermis and prefrontal cortex of individuals with autism. Methods In the current study we employed the mGluR5 tracer [18F]-3-fluoro-5-[(pyridin-3-yl)ethynyl]benzonitrile ([18F]-FPEB) to quantify mGluR5 binding in vivo in adults with autism vs. healthy controls using positron emission tomography (PET). Results We identified significantly higher [18F]-FPEB binding potential in the postcentral gyrus and cerebellum of individuals with autism. There was a significant negative correlation between age and [18F]-FPEB binding potential in the cerebellum but not in the postcentral gyrus. In the precuneus, [18F]-FPEB binding potential correlated positively with the lethargy subscale score for the Aberrant Behavioral Checklist (ABC). In cerebellum, there were significant negative correlations between [18F]-FPEB binding potential and ABC total score, ABC hyperactivity subscale score, and the ABC inappropriate speech subscale score. Conclusions These novel findings demonstrate for the first time that mGluR5 binding is altered in critical brain areas of subjects with autism, suggesting abnormal glutamate signaling in these regions. Finally, the correlations between altered [18F]-FPEB binding potential in the cerebellum and precuneus suggest that some autistic symptoms may be influenced by abnormal glutamate signaling.