Background Mucopolysaccharidosis type III (Sanfilippo syndrome) is a group of four autosomal recessive enzyme deficiencies (A, B, C, and D) leading to tissue accumulation of heparan sulfate. MPS IIIA results from deficiency of N-sulfoglucosamine sulfohydrolase, encoded by the SGSH gene; IIIB from deficiency of N-α-acetylglucosaminidase (encoded by NAGLU); IIIC from deficiency of heparan acetyl-CoA:alpha-glucosaminide N-acetyltransferase (HGSNAT). The primary characteristic of MPS III is the degeneration of the central nervous system resulting in mental retardation and hyperactivity, with initial normal development followed by neurocognitive decline leading to death. We investigated the molecular genetic characteristics of the SGSH gene, NAGLU gene, and HGSNAT gene in Korean MPS III patients. Methods Nineteen patients from 18 families were enrolled in the study. A diagnosis of MPS III was made based on clinical features, disease specific enzyme activity assay and molecular analysis. RESULTS: We found six mutant alleles in six patients with MPS IIIB including four known mutations of c.1444C＞T (p.Arg482Trp), c.200T＞C (p.Leu67Pro), c.1694G＞C (p.Arg565Pro) and c.607C＞T (p.Arg203*). Two were novel variants of unknown significance: c.1976C＞T (p.Ala659Val), and c.775C＞T (p.Gln259*). In eight patients with MPS IIIA, five known mutation alleles were found: c.703G＞A (p.Asp235Asn), c.1040C＞T (p.Ser347Phe), c.544C＞T (p.Arg192Cys), c.69delG (p.Asn24Thrfs*240), and c.1094A＞G (p.Gln365Arg). Three alleles were novel variants of likely pathogenic: c.703G＞C (p.Asp235His), c.812C＞T (p.Thr271Met), and c.1129C＞T (p.Arg377Cys). Only one patient was found to be a compound heterozygote for 2 known mutations of HGSNAT gene (c.234+1G＞A (p.D40Vfs*19) and c.1150C＞T (p.R384*)). The mean age of symptom onset was 3.0 years and the mean age at diagnosis was 6.6 years old. CONCLUSIONS: Of total 14 patients with MPS III diagnosed from molecular analysis, c.1444C＞T (p.Arg482Trp) was a hot spot for MPS IIIB (58.3%) and c.1040C＞T (p.Ser347Phe) for MPS IIIA (37.5%) in Korean patients. In addition to advances in molecular diagnostics, thorough genotype-phenotype correlation can be followed by proper disease prognosis and clinical treatment.