Main conclusionThis DNA fingerprinting test confirmed 195 unique Corylus sp. accessions that were used to build a reference database for identity verification of unknown hazelnut trees from three locations in Ontario.AbstractHazelnut is one of the most profitable tree nuts worldwide. Development of a hazelnut industry in Ontario is urgently required, but economically important cultivars must be genetically verified first in order to meet industry standards. Traditional methods for cultivar identification are largely trait-based and unreliable. In this study, a multiplexed fingerprinting test was modified to allow for hazelnut cultivar discrimination at the DNA level. Fourteen highly polymorphic SSR markers covering the 11 linkage groups of Corylus genome were PCR amplified in multiplex using fluorescent-labelled primers. PCR conditions and primer physical properties were optimized to generate a clear signal for each locus. The 14 SSRs were used to fingerprint 195 unique Corylus accessions collected from the USDA-NCGR. Fragment sizes were subjected to a UPGMA clustering analysis which separated Corylus accessions based on species and geographic origin. For validation purposes, hazelnut leaves from three locations in Ontario were collected for identity verification using this DNA fingerprinting test. As a result, 33.3% of the unknown trees were duplicates of seven distinct genotypes and a small percentage (8.3%) of these were identical to reference Corylus hybrids. These results reflect common mislabelling issues and genotype duplications that can prevent a uniform plant propagation system. Implementation of this test together with the addition of more unique accessions to the reference database will help verification of trueness-to-type of economically important cultivars for the hazelnut industry.