We sampled the 5' end of the granule-bound starch synthase gene (GBSSI or waxy) in Rosaceae, sequencing 108 clones from 18 species in 14 genera representing all four subfamilies (Amygdaloideae, Maloideae, Rosoideae, and Spiraeoideae), as well as four clones from Rhamnus catharticus (Rhamnaceae). This is the first phylogenetic study to use the 5' portion of this nuclear gene. Parsimony and maximum-likelihood analyses of 941 bases from seven complete and two partial exons demonstrate the presence of two loci (GBSSI-1 and GBSSI-2) in the Rosaceae. Southern hybridization analyses with locus-specific probes confirm that all four Rosaceae subfamilies have at least two GBSSI loci, even though only one locus has been reported in all previously studied diploid flowering plants. Phylogenetic analyses also identify four clades representing four loci in the Maloideae. Phylogenetic relationships inferred from GBSSI sequences are largely compatible with those from chloroplast (cpDNA: ndhF, rbcL) and nuclear ribosomal internal transcribed spacer (nrITS) DNA. Large clades are marked by significant intron variation: a long first intron plus no sixth intron in Maloideae GBSSI-1, a long fourth intron in Rosoideae GBSSI-1, and a GT to GC mutation in the 5' splice site of the fourth intron in all GBSSI-2 sequences. Our data do not support the long-held hypothesis that Maloideae originated from an ancient hybridization between amygdaloid and spiraeoid ancestors. Instead, Spiraeoideae genera (Kageneckia and Vauquelinia) are their closest relatives in all four GBSSI clades.