Illumina mRNA-Seq is comparable to microarray analysis for transcript quantification but has increased sensitivity and, importantly, the potential to distinguish between homoeologous genes in polyploids. Using a novel curing process, we adapted a reference sequence that was a consensus derived from ESTs from both Brassica A and C genomes to one containing A and C genome versions for each of the 94,558 original unigenes. We aligned reads from Brassica napus to this cured reference, finding 38% more reads mapping in resynthesised lines and 28% in natural lines. Where the A and C versions differed at single nucleotide positions, termed inter-homoeologue polymorphisms (IHPs), we were able to apportion expression in the polyploid to the A or C genome homoeologues. 43,761 unigenes contained at least one IHP, with a mean frequency of 10.5 per kb unigene sequence. 6,350 of the unigenes with IHPs were differentially expressed between homoeologous gene pairs in resynthesised B. napus. 3,212 unigenes showed a similar pattern of differential expression across a range of natural B. napus crop varieties and, of these, 995 were in common with resynthesised B. napus. Functional classification showed over-representation in gene ontology categories not associated with dosage-sensitivity.