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 were bred and released, but all the qualitative resistance genes released were overcome by pathogen evolution within a short period. In 1994, breakdown of the RMlp7 resistance gene was detected in Belgium and Northern France. New virulent M. larici-populina individuals spread all over Western Europe in less than five years, causing very destructive epidemics, and lead to a complete replacement of the pathogen’s populations. Through a genome-wide association study (GWAS), we identified a locus in the genome of M. larici-populina, that corresponds to the candidate avirulence gene AvrMlp7, whose mutation is responsible for RMlp7 resistance breakdown. To further characterize this effector, we used a population genetics approach on a set of almost 300 individuals collected throughout a 28-year period encompassing the resistance breakdown event. Genotyping at the avirulence locus highlighted two different mutations: a non-synonymous mutation and a complete deletion of this locus. The temporal survey at the candidate locus revealed that both mutations pre-existed long before the breakdown, but at a heterozygous state. Using a reverse ecology approach, we identified the first candidate avirulence gene in this rust fungus.