A sodium azide-mutagenized population of barley (cv. ‘Morex’) was developed and utilized to identify mutants at target genes using the ‘targeting induced local lesions in genomes’ (TILLING) procedure. Screening for mutations at four agronomically important genes (HvCO1, Rpg1, eIF4E and NR) identified a total of 22 new mutant alleles, equivalent to the extrapolated rate of one mutation every 374 kb. All mutations except one were G/C to A/T transitions and several (approximately 68%) implied a change in protein amino acid sequence and therefore a possible effect on phenotype. The high rate of mutation detected through TILLING is in keeping with the high frequency (32.7%) of variant phenotypes observed amongst the M3 families. Our results indicate the feasibility of using this resource for both reverse and forward genetics approaches to investigate gene function in barley and related crops.