TILLING (Targeting Induced Local Lesions IN Genomes), a popular reverse genetics approach in barley research, combines plant mutagenesis with efficient mutation detection for studying biological function of a specific gene. The high mutation frequency within a TILLING population principally enables the identification of induced variations in (almost) all genes of a given species (more precisely a given genotype of a species) of interest, which can be tested for their functional impact on morphological and/or physiological characteristics of the plant. Several TILLING populations induced by chemical mutagenesis were established for barley (Talame et al., Plant Biotechnol J 6:477–485, 2008; Gottwald et al., BMC Res Notes 2:258, 2009; Caldwell et al. Plant J 40:143–150, 2004) and showed the possibility for adapting protocols to develop further populations. This chapter describes a chemical mutagenesis protocol for barley seeds and two independent procedures for efficient single nucleotide polymorphism (SNP) detection in a large number of mutagenized plants either by slab-gel- or capillary gel-based electrophoreses on the LI-COR 4300 DNA Analyzer and the AdvanCE FS96 instruments, respectively.