Coffee plants are seriously affected by leaf rust (Hemileia vastatrix) and loss of resistance to emerging races is a current threat. A deep knowledge of the mechanisms of pathogenicity is necessary for better understanding the plant resistance mechanisms, particularly because the Coffea spp. – H. vastatrix interaction follows the gene for gene theory, enabling the inference of virulence genes in the pathogen and the identification of susceptibility genes in the plant from resistance/susceptibility phenotypes. In the absence of H. vastatrix genome sequence information, transcriptomic analysis is the most effective gene discovery strategy, prompting gene family identification, the establishment of phylogenetic relationships and the development of PCR based molecular markers. High-throughput sequencing of cDNA transcripts by 454 pyrosequencing is capable of producing millions of bases per run, enabling large-scale expression analysis. The objective of this work is the characterisation and comparison of H. vastratrix transcriptomes at three key differentiation/infection stages (germinated uredospores, appressoria and intercellular hyphae with haustoria) for an isolate from race XIV (containing the virulence genes v2,3,4,5). For such, cDNA was obtained from germinated uredospores and appressoria produced in vitro, as well as from intercellular hyphae with haustoria (infected coffee leaves 21 days after inoculation) and subjected to GS-Flex Titanium 454 sequencing (each library comprising over 500000 reads of 300-600bp). Prior to annotation, for the in vivo sample and in the absence of genome sequencing information for both organisms, H. vastatrix transcripts were separated from Coffea sp. transcripts by the analysis of codon usage in ESTs (programme EST3), of GC content, and of blastn homology score against angiosperms and basidiomycetes genomes, as well as of blastx homology score against coffee and Pucciniales EST libraries. The three H. vastatrix EST collections (germinating uredospores, appressoria and intercellular hyphae with haustoria) were annotated using international databases (nr, Swissprot, GO, KOG), aiming to attribute a putative gene function. A particular attention was given to the comparison with the Pucciniales genome sequences (Melampsora larici-populina and Puccinia spp.) and Pucciniales EST databases, in order to identify homologues of genes with relevant function in pathogenicity. The libraries representing the three H. vastatrix differentiation/infection stages were compared among them, in order to identify genes differentially expressed. Genes identified were further studied by RT-qPCR expression analysis in a detailed time-course of H. vastatrix differentiation/infection (Vieira et al., 2010). The identification of H. vastatrix genes involved in pathogenicity will lead to a better understanding of the molecular basis of coffee rust gene for gene interaction.