Background: Congenital hyperinsulinism (CHI) is characterized by an over secretion of insulin by the pancreatic ƒÒ-cells. This condition is mostly caused by mutations in ABCC8 or KCNJ11 genes encoding the SUR1 and KIR6.2 subunits of the ATP-sensitive potassium (KATP) channel. CHI patients are classified according to their responsiveness to diazoxide and to their histopathological diagnosis (either focal, diffuse or atypical forms). Here, we raise the benefits/limits of the genetic diagnosis in the clinical management of CHI patients. Methods: ABCC8/KCNJ11 mutational spectrum was established in 109 diazoxide-unresponsive CHI patients for whom an appropriate clinical management is essential to prevent brain damage. Relationships between genotype and radiopathological diagnosis were analysed. Results: ABCC8 or KCNJ11 defects were found in 82% of the CHI cases. All patients with a focal form were associated with a single KATP channel molecular event. In contrast, patients with diffuse forms were genetically more heterogeneous: 47% were associated with recessively inherited mutations, 34% carried a single heterozygous mutation and 19% had no mutation. There appeared to be a predominance of paternally inherited mutations in patients diagnosed with a diffuse form and carrying a sole KATP channel mutation. Conclusions: The identification of recessively inherited mutations related to severe and diffuse forms of CHI provides an informative genetic diagnosis and allows prenatal diagnosis. In contrast, in patients carrying a single KATP channel mutation, genetic analysis should be confronted with the PET imaging to categorize patients as focal or diffuse forms in order to get the appropriate therapeutic management.