Crucifer downy mildew is caused by the obligatory biotrophic oomycete Hyaloperonospora parasitica (formerly Peronospora parasitica). So far, isolates infecting Arabidopsis thaliana have proven to be non-pathogenic on other crucifers and, despite its unequivocal merit as a research model, the pathosystem A. thaliana-H. parasitica by itself will not provide all the answers onto crucifer downy mildew genetics and biology. In this report, we present the development of a differential display (DD)-based strategy, suitable for high-throughput analysis of expressed sequence tags (ESTs) in plant-pathogen interactions, in this work applied to the analysis of the pathosystem Brassica oleracea-H. parasitica interaction transcriptome. Our purpose was the mining for pathogen-specific ESTs that can be used in future research for virulence factors and Avr genes. A total of 743 specific cDNAs showing differential expression in B. oleracea seedlings infected with H. parasitica, as opposed to healthy seedlings, were isolated by DD-PCR. We found 21 exclusively H. parasitica cDNAs from 433 sequenced DD clones, 18 encoding for potential new genes. Our results reinforce the abilities of DD-PCR for differential screening of pathosystems transcriptomes, leading to the finding of more new potential genes than the previously used techniques. Both the improved DD-based methodology and the graphical representations based on Venn diagrams from polyominoes are appropriate for large-scale analysis of multiple interaction transcriptomes. The obtained data are also innovative since this is the first approach to study the interaction of H. parasitica with its natural host.