Poplar rust, caused by the pathogenic fungus Melampsora larici-populina (Basidiomycota, Pucciniales), is the main phytosanitary constraint for commercial poplar cultivation in Europe and other parts of the world. In the last 50 years, many rust-resistant cultivars have been bred and released, but all the qualitative resistance genes (i.e., major resistance genes) released were overcome by pathogen evolution within a short period. Qualitative resistance is particularly subject to breakdown by pathogen evolution for perennial hosts, such as poplar trees, because of the wide inequality between the pathogen’s rapid generation time and the time needed to deploy new host varieties. For instance, resistance R7 was released in poplar plantations in the 1980s, and some R7 cultivars were widely planted in northwestern Europe. In 1994, breakdown of the R7 resistance was detected in Belgium and northern France, and virulent 7 M. larici-populina individuals spread all over western Europe in less than 5 years, causing very destructive epidemics, and leading to a complete replacement of the pathogen’s populations, on both cultivated and wild poplars. Through a genome-wide association study, we recently identified a locus in the genome of M. larici-populina that likely corresponds to the Avr7 avirulence locus, whose mutation is responsible for R7 resistance breakdown in poplar. The candidate avirulence gene possesses some characteristics of canonical effectors (coding a small cysteine-rich protein, specific to M. larici-populina, early expressed and of unknown function) but lacks a secretion signal. Our aim is to functionally validate this avirulence locus and study its evolution over time in natural populations of the pathogen. This would be the first avirulence gene identified in this biotrophic fungal species. A better understanding of the evolution of the pathogen’s genome and the genetic architecture of virulence would help in defining breeding strategies for durable resistance, especially for perennial crops, such as poplars.