Fragile X testing is a priority in the evaluation of autism spectrum disorders (ASD) cases because identification of the FMR1 mutation leads to new treatment options. This study is focused on determining the prevalence of the FMR1 gene mutation among ASD cases in Indonesia. DSM-IV-TR criteria were administered to diagnose ASD; symptom severity was classified using the Childhood Autism Rating Scale. Cytogenetic analysis, polymerase chain reaction, and Southern blot for FMR1 gene analysis were carried out to confirm the diagnosis of fragile X syndrome. The fragile X site and FMR1 full mutation allele were identified in 3 out of 65 (4.6%) and 4 out of 65 (6.15%) children aged 3–17 years (57 boys and 8 girls), respectively. The Fragile X laboratory workup is essential in the evaluation of patients with ASD. Molecular analysis is most accurate, while cytogenetic documentation of the fragile X site can also be useful if molecular testing is not available.