Crop germplasm conserved in genebanks, are a fundamental resource of genetic diversity for crop improvement activities, underpinning future food security and sustainable agricultural practices. However, taxonomic errors in genebank germplasm (due to misclassification, contamination and poor data collation) restrict the effective use of this material for correct purpose. Earlier studies investigating species genetic diversity using genebank germplasm, have shown varying levels of taxonomic error within the Brassica species. In response to this reported taxonomic error of global collections, together with the availability of a multiplex PCR (MPCR) marker, targeting the specific chromosomes (A, B and C) of the six Brassica species in U's triangle, this study was undertaken to confirm the taxonomic identity of accessions within the Australian Grains Genebank's (AGG) long-term Brassica collection. A total of 5161 accessions were analysed with MPCR for taxonomic identification, of which, 4842 (93.8%) were confirmed to be consistent (correct) with their labelled taxonomy, while the remaining 319 (6.2%) were identified as taxonomically inconsistent (in-error). Through the evaluation of earlier regeneration and original seed of the error accessions with MPCR, we determined that 80.9% of the taxonomic errors were traced back to the original seed, while 19.1% of errors were the result of genebank seed regeneration handling practices. Results from this study directly enhance information of the AGG Brassica collection and shape directions for distribution, acquisition and regeneration practices within the AGG and potentially other global genebanks, which will facilitate in a more effective use of these valuable genetic resources by researchers and breeders.