Monogenic diabetes (MD) is a rare form of diabetes caused by a defect in a single gene, and patients over 25 years of age are often overlooked despite the value of genetic diagnosis for personalized treatment. The study evaluated genetic sequencing in 509 patients with suspected MD from 2017–2024 in all age groups, confirming a molecular diagnosis in 78 individuals (15.3%). Testing was performed in patients with negative pancreatic autoantibodies, a family history of diabetes, or stable hyperglycemia without the need for insulin. The median age at diagnosis of MD was 18.3 years (range 4–68.1), with a median duration of diabetes of 4.5 years (0–50). Forty-three patients (55.1%) were diagnosed before 25 years and 35 (45.6%) after 25 years; in both groups, GCK variants predominated (81.4% and 74.3%), followed by HNF1A, HNF4A and HNF1B. After confirmation of diagnosis, 75% (18 of 24) of eligible patients changed treatment according to genotype, while 6 did not benefit from adjustment. The findings show a high diagnostic yield and the need to expand the testing criteria for gene-directed therapy.