The allelic compositions of high- and low-molecular-weight subunits of glutenins (HMW-GS and LMW-GS) among European spelt (Triticum spelta L.) and related hexaploid and tetraploid Triticum species were investigated by one- and two-dimensional polyacrylamide-gel electrophoresis (PAGE) and capillary electrophoresis (CE). A total of seven novel glutenin alleles (designated A1a*, B1d*, B1g*, B1f*, B1j*, D1a* at Glu-1 and A3h at the Glu-3 loci, respectively) in European spelt wheat were detected by SDS-PAGE, which were confirmed further by employing A-PAGE and CE methods. Particularly, two HMW-GS alleles, Glu-B1d* coding the subunits 6.1 and 22.1, and Glu-B1f* coding the subunits 13 and 22*, were found to occur in European spelt with frequencies of 32.34% and 5.11%, respectively. These two alleles were present in cultivated emmer (Triticum dicoccum), but they were not observed in bread wheat (Triticum aestivum L.). The allele Glu-B1g* coding for 13* and 19* subunits found in spelt wheat was also detected in club wheat (Triticum compactum L.). Additionally, two alleles coding for LMW-GS, Glu-A3h and Glu-B3d, occurred with high frequencies in spelt, club and cultivated emmer wheat, whereas these were not found or present with very low frequencies in bread wheat. Our results strongly support the secondary origin hypothesis, namely European spelt wheat originated from hybridization between cultivated emmer and club wheat. This is also confirmed experimentally by the artificial synthesis of spelt through crossing between old European emmer wheat, T. dicoccum and club wheat, T. compactum.