A study was carried out to assess the utility of the Illumina sequencing platform for the identification of new variation in the available B. napus DH12075 EMS mutant population (http://www.botany.ubc.ca/can-till/). The approach utilizes multi-dimensional pooling of extracted genomic DNA from a portion of the mutant population to screen for variant nucleotides. Overlapping amplicons were generated to cover target B. napus genes, then the amplicons were pooled and barcoded to allow for sufficient resolution of potential mutant lines. Sequencing reads were partitioned according to the barcodes, each pool was subsequently aligned to the reference gene sequence and mutation frequencies at each base were calculated from reads with high Phred base quality scores. The lower frequency of EMS mutations in the pool is easily distinguishable from the much higher frequency of SNPs at A and C genome homoeologous loci or hemi-SNPs making this a robust approach for mutation detection in B. napus. We were able to validate mutations previously characterized by TILLING as well as identify additional mutations not previously detected by TILLING. This approach combined with increases in sequencing throughput may be utilized to detect mutations in larger targeted regions or indeed whole exomes for the entire DH12075 EMS mutant population to create a valuable long-term resource.