Brassica genomes are relatively large and complex due to historic duplication events, the amplification of families of transposable elements, and polyploidisation. The development of second generation DNA sequencing methods is rapidly changing plant genome research and we are applying this technology for the analysis of the Brassica genomes. We have generated genome sequence data for several Brassica species and developed tools for the analysis of this data. These tools can be applied for gene and molecular marker discovery, providing an unprecedented insight into genome structure and variation to support Brassica crop improvement. Single Nucleotide Polymorphisms (SNPs) are the most abundant genetic marker and predominate applications in modern plant genetic analysis, such as diversity analysis, genetic trait mapping, association studies, and marker assisted selection. SNPs are direct markers, as the sequence information provides the exact nature of the allelic variants. Furthermore, this sequence variation can have a major impact on how the organism develops and responds to the environment. Here we present the discovery of SNPs from amphidiploid Brassica species using Illumina GAIIx and HiSeq2000 sequence data. More than 1 million SNPs have been identified across eight varieties of B. napus. The number of predicted SNPs was distributed evenly across the 19 chromosomes, with variation as expected according to chromosome length. The SNP base changes were recorded. SNP density varied across the chromosomes suggesting evidence of selection.The identified SNPs have been used for the development of a 6K Illumina Infinium assay.