Isolated methylmalonic aciduria (MMA) is an autosomal‐recessive disorder of propionate metabolism that is most commonly caused by mutations in the methylmalonyl‐CoA mutase (MUT) gene (mut‐type MMA). We investigated a cohort of 151 patients, classifying 114 patients as mut⁰ and 32 as mut⁻ (five not defined). As per the definition, mut⁻ patients showed a higher propionate incorporation ratio in vitro, which was correlated to a considerably later age of onset compared with mut⁰ patients. In all patients, we found a total of 110 different mutations, of which 41 were novel. While the missense alleles p.Asn219Tyr, p.Arg369His, and p.Arg694Trp recurred in >10 alleles, 47 mutations were identified only once, suggesting many patients carry private mutations. Deficient alleles in the mut⁻ subclass were almost exclusively caused by missense mutations, found disproportionately in the C‐terminal cofactor binding domain. On the contrary, only half of the mut⁰ mutations were of the missense type. Western blot analysis revealed reduced MUT protein for all 34 cell lines (27 mut⁰, seven mut⁻) tested, suggesting protein instability as a major mechanism of deficiency in mut‐type MMA. This large‐scale evaluation helps to characterize the landscape of MUT mutations and their relationship to dysfunction and disease.