Classical galactosemia is an autosomal recessive disorder of galactose metabolism due to galactose-1-phosphate uridyltransferase (GALT) deficiency. Treatment through restriction of dietary galactose intake is lifesaving, but, in spite of this diet, most patients develop abnormalities. In this paper we report the mutational spectrum of classical galactosemia in a cohort of 123 Dutch patients, all with biochemically proven classical galactosemia. In the human GALT gene, which is located on chromosome 9p13, we identified 24 different mutations, including nine mutations that have not been reported previously. The novel mutations include five missense mutations (c.152G>A/p.R51Q, c.404C>T/p.S135W, c.687G>T/p.K229N, c.756G>T/p.Q252H, and c.1140A>C/p.X380C), a frame shift mutation (c.410dupT), a splice site mutation (c.821-2A>G), a possible branch point mutation (c.508-29delT), and a large deletion encompassing at least exons 1-11. Six of these novel mutations were found in patients of Dutch descent: p.R51Q, p.S135W, p.K229N, p.Q252H, p.X380C, and c.410dupT.