AbstractIn 2016, Methylation-Specific Quantitative Melt Analysis (MS-QMA) on 3,340 male probands increased diagnostic yield from 1.60% to 1.84% for fragile X syndrome (FXS) using a pooling approach. In this study probands from Lineagen (UT, U.S.A.) of both sexes were screened using MS-QMA without sample pooling. The cohorts included: (i) 279 probands with no FXS full mutation (FM: CGG > 200) detected by AmplideX CGG sizing; (ii) 374 negative and 47 positive controls. MS-QMA sensitivity and specificity in controls approached 100% for both sexes. For male probands with no FM detected by standard testing (n = 189), MS-QMA identified abnormal DNA methylation (mDNA) in 4% normal size (NS: < 44 CGGs), 6% grey zone (CGG 45–54) and 12% premutation (CGG 54–199) alleles. The abnormal mDNA was confirmed by AmplideX methylation sensitive (m)PCR and EpiTYPER tests. In contrast, no abnormal mDNA was detected in 89 males with NS alleles from the general population. For females, 11% of 43 probands with NS alleles by the AmplideX sizing assay had abnormal mDNA by MS-QMA, with FM / NS mosaicism confirmed by AmplideX mPCR. FMR1 MS-QMA analysis can cost-effectively screen probands of both sexes for methylation and FM mosaicism that may be missed by standard testing.