Introduction: Fragile X syndrome (FXS) is one of the most prevalent genetic causes of developmental disability, representing the most frequent form of inherited severe cognitive deficit. The present study was undertaken to investigate FXS and its prevalence in moderate mentally retarded people in patients. Materials and methods: Nineteen people with moderate mental retardation (MR) who were clinically suspicious to have FXS were screened for FXS by using cytogenetic and molecular methods. Blood samples were collected and cultured in specific culture media. G-Banding method was used for karyotyping. To ensure correct results of cytogenetic testing, four suspected case of FXS were tested by PCR. Results were analyzed using logistic regression analysis. Results: Four patients (4%) were found to express fragile X site at q27.3. The results showed that the relationship of FXS with familial, economic status was not significant, but the relationship of FXS with MR and family history was significant. Conclusion:The frequency of FXS positive cases found in this study is similar to other reports of FXS in preselected patients.