Sotos syndrome is an overgrowth condition predominantly caused by truncating mutations, missense mutations restricted to functional domains, or deletions of NSD1. NSD1 is a member of a protein family that includes NSD2 and NSD3, both of which show 70-75% sequence identity with NSD1. This strong sequence similarity suggests that abrogation of NSD2 or NSD3 function may cause non-NSD1 Sotos cases or other overgrowth phenotypes. To evaluate this hypothesis, we mutationally screened NSD2 and NSD3 in 78 overgrowth syndrome cases in which NSD1 mutations and deletions had been excluded. Additionally, we used microsatellite markers within the vicinity of the genes to look for whole gene deletions. No truncating mutations or gene deletions were identified in either gene. We identified two conservative missense NSD2 alterations in two non-Sotos overgrowth cases but neither was within a functional domain. We identified three synonymous and two intronic variants in NSD2 and two synonymous base substitutions in NSD3. Our results suggest that despite strong sequence similarity between NSD1, NSD2 and NSD3, the latter genes are unlikely to be making a substantial contribution to overgrowth phenotypes and thus may operate in distinct functional pathways from NSD1.