Maturity-onset diabetes of the young (MODY) is an uncommon monogenic type of diabetes mellitus. Detecting genetic biomarkers for MODY is a necessity for precise diagnosis and treatment. The majority of MODY genetic predisposition has been documented in European populations and a lack of information is present in Iranians which leads to misdiagnosis as a consequence of defects in unknown variants. In this study, using genetic variant information of 20,002 participants from the family-based TCGS (Tehran cardiometabolic genetic study) cohort, we evaluated the genetic spectrum of MODY in Iran. We concentrated on 14 previously discovered and two novel MODY-causing genes (RFX6 and NKX6-1) that were thought to be responsible for MODY. To find disease-causing mutations, genetic variants were evaluated for their pathogenicity. We discovered 6 mutations that were previously reported in the ClinVar as pathogenic/likely pathogenic (P/LP) for MODY in 45 participants from 24 families. Three potentially novel MODY-causing mutations were also identified, which showed complete disease penetrance (100%) in 10 subjects from 5 families. This is the first family-based study to define the genetic spectrum and estimate the prevalence of MODY in the Iran. The newly discovered mutations need to be investigated by additional studies.