Mucopolysaccharidoses (MPSs) are rare, heterogeneous inborn errors of metabolism (IEM) diagnosed through a combination of clinical, biochemical, and genetic investigations. The aim of this study was molecular characterization of the largest cohort of Iranian MPS patients (302 patients from 289 unrelated families), along with tracking their ethnicity and geographical origins. 185/289 patients were studied using an IEM-targeted NGS panel followed by complementary Sanger sequencing, which led to the diagnosis of 154 MPS patients and 5 non-MPS IEMs (diagnostic yield: 85.9%). Furthermore, 106/289 patients who were referred with positive findings went through re-analysis and confirmatory tests which confirmed MPS diagnosis in 104. Among the total of 258 MPS patients, 225 were homozygous, and 9 had copy number variations, and 90 had novel variants. MPS IV was the most common type (34.8%) followed by MPS I (22.7%) and MPS VI (22.5%). Geographical origin analysis unveiled a pattern of distribution for frequent variants in ARSB (c.430G>A, c.962C>T, c.281C>A), GALNS (c.319G>A, c.860C>T, c.1042A>G), and IDUA (c.1A>C, c.1598C>G). Our extensive patient cohort reveals the genetic and geographic landscape of MPS in Iran, which provides insight into the genetic epidemiology of MPS and can facilitate a more cost-effective, time-efficient diagnostic approach based on the region-specific variants.