Sotos syndrome is a rare genetic disorder caused by haploinsufficiency of the NSD1 (nuclear receptor binding SET domain containing protein 1) gene. No clinical diagnostic consensus criteria are published yet, and molecular analysis reduces the clinical diagnostic uncertainty. We screened 1530 unrelated patients enrolled from 2003 to 2021 at Galliera Hospital and Gaslini Institute in Genoa. NSD1 variants were identified in 292 patients including nine partial gene deletions, 13 microdeletions of the entire NSD1 gene, and 115 novel intragenic variants never previously described. Thirty-two variants of uncertain significance (VUS) out of 115 identified were re-classified. Twenty-five missense NSD1 VUS (25/32, 78.1%) changed class to likely pathogenic or likely benign, showing a highly significant shift in class (p < 0.01). Apart from NSD1, we identified variants in additional genes (NFIX, PTEN, EZH2, TCF20, BRWD3, PPP2R5D) in nine patients analyzed by the NGS custom panel. We describe the evolution of diagnostic techniques in our laboratory to ascertain molecular diagnosis, the identification of 115 new variants, and the re-classification of 25 VUS in NSD1. We underline the utility of sharing variant classification and the need to improve communication between the laboratory staff and the referring physician.