Background Fragile X syndrome (FXS) is the most common form of inherited intellectual disability, caused by CGG-repeats expansion (> 200 repeats). Premutation alleles (PM) (55–200 CGG repeats) are associated with tremor ataxia syndrome (FXTAS), fragile X-associated primary ovarian insufficiency (FXPOI), and autistic problems. Aim To screen the frequency of premutation carriers using molecular diagnostic assays, in a cohort of Egyptian males with suspected clinical features of (FXS) checking for the presence of premutation alleles. Methods The current study comprised 192 Egyptian male children, 92 participants presented with intellectual disability, delayed language development, autistic-like features, behavioral difficulties, anxiety, seizures, and depression compared to 100 healthy males. All cases were subjected to clinical and neuroimaging assessments, when indicated as well as molecular analysis using methylation-specific PCR (MS-PCR) and quantitative real-time PCR (qRT-PCR). Results Thirty-four premutation carriers out of 92 Egyptian males (37%) of CGG repeats (55 to 200) were illustrated with elevated FMR1 mRNA expression level (p-value < 0.001). Additionally, 2 intermediate (IM) cases (0.03%) (45–55 CGG repeats) showed poor increase in expression level (p-value = 0.02838) plus 6 full mutation (FM) patients (0.07%) with (> 200 CGG repeats) (p-value < 0.001) resulted in FMR1 gene silence. Conclusion Molecular diagnostic assay including (MS-PCR) and (qRT-PCR) proved to be a sensitive and rapid screening tool for the detection of premutation cases. Furthermore, the presence of positive correlation between FMR1 mRNA expression levels with CGG repeats in premutation cases could serve as a potential diagnostic marker. Application of these diagnostic tools on larger number clinically suspected cases is recommended.