© The Author(s) 2016. Mutations in the MECP2 gene cause Rett syndrome (RTT). MeCP2 binds to chromocentric DNA through its methyl CpG-binding domain (MBD) to regulate gene expression. In heterozygous females the variable phenotypic severity is modulated by non-random X-inactivation, thus making genotype-phenotype comparisons unreliable. However, genotype-phenotype correlations in males with hemizygousMECP2 mutations can provide more accurate insights in to the true biological effect of specific mutations. Here, we compared chromatin organization and binding dynamics for twelve MeCP2 missense mutations (including two novel and the five most common MBD missense RTT mutations) and identifiedacorrelation with phenotype in hemizygous males. We observed impaired interaction of MeCP2-DNA for mutations around the MBD-DNA binding interface, and defective chromatin clustering for distal MBD mutations. Furthermore, binding and mobility dynamics show a gradient of impairment depending on the amino acid properties and tertiary structure within the MBD. Interestingly, a wide range of phenotypic/clinical severity, ranging from neonatal encephalopathy to mild psychiatric abnormalities were observed and all are consistent with our functional/molecular results. Overall, clinical severity showed a direct correlation with the functional impairment of MeCP2. These mechanistic and phenotypic correlations of MeCP2 mutations will enable improved and individualized diagnostics, and may lead to personalized therapeutic interventions.