Leaf rust (Hemileia vastatrix), the main disease of Arabica coffee, causes up to 30% yield losses in susceptible plants. Although resistant genotypes are identified against all known rust races, loss of resistance to emerging races is a current threat. A thorough knowledge of the plant resistance mechanisms requires a deep understanding of the mechanisms of pathogenicity, 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. However, very little is known on the molecular mechanisms governing the fungal infection process. Addressing Coffea spp. - H. vastatrix interactions from the pathogen’s perspective, this work aims to study H. vastatrix genes expressed during the infection process in coffee leaves. For such, an in vitro system was used to obtain the first steps of H. vastatrix differentiation process, while the post-penetration infection stages were obtained in planta. Uredospores germination and appressoria differentiation were monitored by light microscopy and no significant differences were observed between artificial membranes and leaves. Nuclear condition of spores, germlings and appressoria observed in vivo and in vitro, studied by DAPI detection, was identical. Infection stages from 24 hours (formation of infection hyphae) to 21 days after inoculation (profuse host tissue colonization) were collected in planta and monitored by light microscopy. Genes described in the literature involved in the differentiation and pathogenicity of other rust fungi were compared with a H. vastatrix EST database, enabling the selection of homologue H. vastatrix candidate genes for differential expression analysis by RT-qPCR during differentiation and infection. These included genes involved in signalling (mitogen-activated protein kinase and Gα protein subunit), transport (aminoacid and sugar), carbohydrate metabolism (manitol dehydrogenase, invertase and chitin deacetylase) and maintenance of the biotrophic interaction (RTP1). The characterisation of the expression patterns of genes involved in the infection process will enhance our comprehension on the genetic basis of pathogenicity in H. vastatrix. Together with the information on the genetics of Coffea spp. resistance to H. vastatrix, this knowledge will improve our understanding on gene for gene interaction in the H. vastatrix – Coffea spp. pathosystem, leading to a more informed deployment of resistant varieties in coffee crops in order to attain a sustainable agriculture.