Potato can be used as a source of modified starches for culinary and industrial processes, but its allelic diversity and tetraploid genome make the identification of novel alleles a challenge, and breeding such alleles into elite lines is a slow and difficult process. An efficient and reliable strategy has been developed for the rapid introduction and identification of new alleles in elite potato breeding lines, based on the ethylmethanesulphonate mutagenesis of dihaploid seeds. Using the granule-bound starch synthase I gene (waxy) as a model, a series of point mutations that potentially affect gene expression or enzyme function was identified. The most promising loss-of-function allele (waxy(E1100)) carried a mutation in the 5'-splice donor site of intron 1 that caused mis-splicing and protein truncation. This was used to establish elite breeding lineages lacking granule-bound starch synthase I protein activity and producing high-amylopectin starch. This is the first report of rapid and efficient mutation analysis in potato, a genetically complex and vegetatively propagated crop.