The genetic diversity of cultivated spelt (Triticum aestivum ssp. spelta) presently is narrow. Evaluation of germplasm collections of spelt on quality level supplemented with DNA analysis is, therefore, of great importance. This study was designed to help the evaluation process for the selection of new spelt varieties with a support of molecular characterization. A total of 30 genotypes, including two common wheat varieties, were included in the evaluation of genetic diversity on quality and DNA levels. According to the quality attributes, spelt flours exhibited medium rheological parameters and many of them had average gluten quality. AFLP analysis was conducted to evaluate phylogenetic relationships and the genetic diversity present in the accessions. A high level of genetic diversity was revealed by the very high PIC values. Two main clusters could be separated on the dendrogram: a cluster with genotypes that have common wheat in their pedigree and another cluster consisting of pure spelt accessions. The extent of genetic diversity in the spelt germplasm collections was confirmed not only by molecular markers but on the basis of quality assessment.