The identification of genetic variation using molecular markers is fundamental to modern plant breeding and research. The present study was undertaken to develop a resource of informative single nucleotide polymorphism (SNP) markers for molecular characterisation of Australian barley germplasm. In total, 190 SNP markers were developed and characterised using 88 elite barley lines and varieties, sampling genetic diversity relevant to Australian breeding programs, and a core set of 48 SNPs for distinguishing among the barley lines was identified. The utility of the core 48-SNP set for distinguishing barley lines and varieties using DNA extracted from grain samples was also assessed. Finally, the 48 SNPs in the core set were converted into simple PCR markers to enable co-dominant SNP genotyping on agarose gel. The SNP markers developed, and in particular the core 48-SNP set, provide a useful marker resource for assessing genetic relationships between individuals and populations of current Australian barley germplasm. They are also useful for identity and purity testing of inbred lines in research, breeding, and commercial applications.