Abstract Background Recent investigations suggest a possible common genetic background between Autism Spectrum Disorders (ASD) and Celiac Disease (CD). However, studies regarding this association are scarce and often limited by the small sample sizes and/or large heterogeneity among ASD groups in terms of demographic and clinical features. The present study aims to investigate the overall CD prevalence (biopsy proven-CD patients plus screening detected tTG and EMA positive cases) in a large population of pre-schoolers with ASD referred to a tertiary care University Hospital. Methods We retrospectively collected data about 382 children (mean age: 46.97 ± 13.55 months; age-range: 18-72 months) consecutively diagnosed as ASD (according to the Diagnostic and Statistical Manual of Mental Disorders 4th edition criteria) over the period 2010–2013, and who performed a serological CD screening. Results The overall CD prevalence was 2.62%, which is statistically significant higher to that reported in the Italian paediatric population ( p = 0.0246). Half of these children had no symptoms or risk factors related to CD when they performed the serological screening. Conclusions If replicated, these data suggest the importance of regular screening for CD in young patients with ASD, and are of relevance for clinical and public health.