The most frequently used method to identify mutations induced by a commonly used mutagen, EMS (ethyl methane sulfonate), in Arabidopsis thaliana has been map-based cloning. The first step of this method is crossing a mutant with a plant of another accession as it requires polymorphisms between accessions for linkage analysis. Therefore, to perform the method routinely, it is greatly preferred to use accession combinations between which enough polymorphisms are already known. Further, it requires laborious examination of a large number of F₂ recombinants using many markers to detect each polymorphism. After linkage analysis narrows down the chromosomal region containing the causal mutation, sequencing candidate genes one by one within the region is necessary until the mutation is finally identified. Overall, this method is generally time-consuming and labor intensive, and it becomes harder when multiple loci are involved in phenotypes. A few recent reports showed that causal mutations induced by EMS could be identified by deep-sequencing technologies with less labor compared with the conventional method when mutants were generated in the Arabidopsis reference Columbia background whose genome organization is well known. Here we report that we succeeded in rapid identification of EMS-induced causal mutations in a non-reference accession background, whose whole genome sequence is not publicly available, using one round of whole genome sequencing. Moreover, in our case, we could monitor the causal locus and the transgenic reporter locus simultaneously, implying that this methodology could theoretically be applicable to analyzing even complex traits. We describe the pipeline of this methodology and discuss its characteristics.