The present study intends to assess H. vastatrix population genetic diversity and differentiation, migration/dispersal patterns and gene flow among populations using a comprehensive and across-time coverage of isolates from different geographical origins, as well as to get some insights on the pathogen evolution. Here we report the initial stage of this study with the analysis of a first set of 31 H. vastatrix isolates from 12 coffee-growing countries using AFLPs and gene sequences. Several loci, including the universally used rDNA-ITS region, β-tubulin 1, TEF1 and candidate genes, were tested for marker informative value. Some level of variability could be detected in ITS and β-tubulin 1 loci providing some insights on the discrimination among H. vastatrix populations and a first step to better understand the underlying population structure. On the other hand, among a set of AFLP selective primers tested for sample screening, two AFLP primer combinations generated distinctive fragment patterns among different isolates. Based on both datasets, population structure and diversity parameters will be discussed, as well as inferences on the spatial distribution of the genetic variability. These first results open the way to unravel the molecular differentiation and the dynamics of H. vastarix populations.