Emerging next-generation sequencing (NGS) technologies are powerful tools for the identification of causal mutations underlying phenotypes of interest in Arabidopsis thaliana. Based on a methodology termed bulked segregant analysis (BSA), whole-genome sequencing data are derived from pooled F2 segregants after crossing a mutant to a different polymorphic accession and are analyzed for single nucleotide polymorphisms (SNPs). Then, a genome region spanning the causal mutation site is narrowed down by linkage analysis of SNPs in the accessions used to produce the F1 generation. Next, candidate SNPs for the causative mutation are extracted by filtering the linked SNPs using multiple appropriate criteria. Effects of each candidate SNP on the function of the corresponding gene are evaluated to identify the causal mutation, and its validity is then confirmed by independent criteria. This chapter describes the identification by NGS analysis of causal recessive mutations derived from EMS mutagenesis.