Background <p>Fragile X syndrome (FXS) is primarily caused by the expansion of CGG repeats in the 5’ untranslated region of the <i>FMR1</i> gene. Accurate detection of expanded <i>FMR1</i> alleles is essential for timely diagnosis and management. Triplet-repeat primed PCR is the most widely used method for detecting FXS; however, it has limitations in detecting low DNA input (&lt; 10&#xa0;ng/μL) and low-level mosaicism (&lt; 5%). This study aimed to develop an improved method for detecting <i>FMR1</i> CGG repeat expansions, outperforming existing methods in efficiency, reliability and sensitivity.</p> Methods <p>We developed a novel four-primer PCR with capillary electrophoresis assay (FP-PCR/CE) and validated its performance in identifying and sizing <i>FMR1</i> alleles using DNA standards and multi-center clinical samples (<i>N</i> = 1690). Comparative analyses were performed against the AmplideX <i>FMR1</i> PCR/CE assay and Southern blot to assess the accuracy, sensitivity, and clinical reliability of this assay.</p> Results <p>The FP-PCR/CE assay demonstrated 100% concordance with DNA standards for CGG repeat sizing and mosaicism detection. It detected DNA input ≥ 2.5&#xa0;ng/μL and mosaic alleles at a mass fraction as low as 1%. In clinical validation, FP-PCR/CE achieved 100% concordance in <i>FMR1</i> allele characterization with both the AmplideX assay and Southern blot, while exhibiting higher sensitivity for detecting mosaicism. Additionally, the assay identified AGG interruptions within <i>FMR1</i> alleles. The FP-PCR/CE assay also reduced testing time to under 7&#xa0;h and lowered the cost to &lt; $80 per test.</p> Conclusions <p>The FP-PCR/CE assay is a rapid, accurate, and cost-effective method for <i>FMR1</i> CGG repeat analysis, offering improved sensitivity for mosaicism detection. Its scalability and reliability support its potential for broader use in FXS carrier screening, clinical diagnosis and research.</p> Graphical abstract <p></p>