Mucopolysaccharidosis (MPS) disorders are heterogeneous and caused by deficient lysosomal degradation of glycosaminoglycans, resulting in distinct but sometimes overlapping phenotypes. Molecular analysis was performed for a total of 355 MPS patients with MPSI (n = 15), MPSII (n = 218), MPSIIIA (n = 86), MPSIIIB (n = 20), MPSIVA (n = 6) or MPSVI (n = 10). This analysis revealed 104 previously unreported mutations: seven in IDUA (MPSI), 61 in IDS (MPSII), 19 in SGSH (MPSIIIA), 11 in NAGLU (MPSIIIB), two in GALNS (MPSIVA) and four in ARSB (MPSVI). The intergenic comparison of the mutation data for these disorders has revealed interesting differences. Whereas IDUA, IDS, NAGLU and ARSB demonstrate similar levels of mutation heterogeneity (0.6-0.675 different mutations per total alleles), SGSH and GALNS have lower levels of mutation heterogeneity (0.282 and 0.455, respectively), due to more recurrent mutations. The type of mutation also varies significantly by gene. SGSH, GALNS and ARSB mutations are usually missense (76.5 %, 81.8 % and 85 %), while IDUA has many more nonsense mutations (56 %) than the other genes (≤20%). The mutation spectrum is most diverse for IDS, including intergenic inversions and multi-exon deletions. By testing 102 mothers of MPSII patients, we determined that 22.5 % of IDS mutations are de novo. We report the allele frequency of common mutations for each gene in our patient cohort and the exonic distribution of coding sequence alterations in the IDS, SGSH and NAGLU genes, which reveals several potential "hot-spots". This further molecular characterization of these MPS disorders is expected to assist in the diagnosis and counseling of future patients.