Mucopolysaccharidoses (MPS) type III known as Sanfilippo syndrome, which is a multisystem lysosomal storage disorder that is characterized by central nervous system degeneration and extreme clinical variability. There are four types of MPS III (A, B, C, and D). All of them are autosomal recessive diseases. Each type is associated with a specific enzyme deficiency that catabolizes the heparan sulfate macromolecule and presents with a wider range of symptoms, including both mental and physical. To the best of our knowledge, there have been no molecular studies conducted on MPS III disease in Oman, and the genetic mutation causing MPS III subtypes remains to be elucidated. Moreover, there is limited knowledge about founder mutations, genotypephenotype correlations, and the functional effect of these mutations on corresponding proteins. Therefore, this project aimed at identifying MPS III causal mutations in Omani patients, functional characterization of causal mutations, understand the phenotypegenotype correlation, and establish a local targeted panel for MPS III patients. Thirteen patients with MPS III, diagnosed from January 2011 to December 2021 at the Royal Hospital, were enrolled. The whole exome sequencing (WES) approach was used to identify causal mutations. Identified variants were validated using sanger sequencing, and characterized using bioinformatics tools and databases. We identified and characterized six mutations in Omani patients with Sanfilippo syndrome. Three of them were novel. In the SGSH gene, two mutations, c.1175delT (p.Phe392Serfs*21) and c.48C>T, were associated with MPS IIIA. In cases with MPS IIIB, three mutations were identified in the NAGLU gene (c.239C>T (p. Ser80Phe), c.746T>A (p.Val249Asp), and c.1669_1670insAGCAGGTCCA, (p.Arg557Glnfs*24). In the MPS IIIC patient, a novel mutation (c.835G>A, p. Asp279Asn) was identified in exon 9 of the HGSNAT gene. Notably, all associated variants were homozygous, which suggested mutation inheritance from a common ancestor. In fact, patients sharing the same variant belong to the same family, tribe, or region. The identified variants are associated with a wide spectrum of clinical phenotypes that may to an extend explain disease heterogeneity. Furthermore, a local diagnostic and prognostic panel for MPS III was established. Finally, we hope the data generated from this project will be used for understanding disease heterogeneity, and selecting an appropriate therapeutic approach.