Determining the clinical significance of germline and somatic KMT2D missense variants (MVs) in Kabuki syndrome (KS) and cancers can be challenging. We analysed 1920 distinct KMT2D MVs that included 1535 germline MVs in controls (Control-MVs), 584 somatic MVs in cancers (Cancer-MVs) and 201 MV in individuals with KS (KS-MVs). The proportion of MVs likely to affect splicing was significantly higher for Cancer-MVs and KS-MVs than in Control-MVs (p = 0.000018). Our analysis identified significant clustering of Cancer-MVs and KS-MVs in the PHD#3 and #4, RING#4 and SET domains. Areas of enrichment restricted to just Cancer-MVs (FYR-C and between amino acids 3043-3248) or KS-MVs (coiled-coil#5, FYR-N and between amino acids 4995-5090) were also found. Cancer-MVs and KS-MVs tended to affect more conserved residues (lower BLOSUM scores, p < 0.001 and p = 0.007). KS-MVs are more likely to increase the energy for protein folding (higher ELASPIC ∆∆G scores, p = 0.03). Cancer-MVs are more likely to disrupt protein interactions (higher StructMAn scores, p = 0.019). We reclassify several presumed pathogenic MVs as benign or as variants of uncertain significance. We raise the possibility of as yet unrecognised 'non-KS' phenotype(s) associated with some germline pathogenic KMT2D MVs. Overall, this work provides insights into the disease mechanism of KMT2D variants and can be extended to other genes, mutations in which also cause developmental syndromes and cancer.