Oilseed rape (Brassica napus) was selected as an example of a polyploid crop, and the Solexa sequencing system was used to generate approximately 20 million expressed sequence tags (ESTs) from each of two cultivars: Tapidor and Ningyou 7. A methodology and computational tools were developed to exploit, as a reference sequence, a publicly available set of approximately 94 000 Brassica species unigenes. Sequences transcribed in the leaves of juvenile plants were aligned to approximately 26 Mb of the reference sequences. The aligned sequences enabled the detection of 23 330–41 593 putative single nucleotide polymorphisms (SNPs) between the cultivars, depending on the read depth stringency applied. The majority of the detected polymorphisms (87.5–91.2%) were of a type indicative of transcription from homoeologous genes from the two parental genomes within oilseed rape, and are termed here ‘hemi-SNPs’. The overall estimated polymorphism rate (~0.047%–0.084%) is consistent with that previously observed between the cultivars analysed. To demonstrate the heritability of SNPs and to assess their suitability for applications such as linkage map construction and association genetics, approximately nine million ESTs were generated, using the Solexa system, from each of four lines of a doubled haploid mapping population derived from a cross between Tapidor and Ningyou 7. Computational tools were developed to score the alleles present in these lines for each of the potential SNPs identified between their parents. For a specimen region of the genome analysed in detail, segregation of alleles largely, although not entirely, followed the pattern expected for genomic markers.