The deployment of plant varieties carrying resistance (R) genes exerts strong selection pressures towards pathogen populations. Several studies reported the rapid evolution of avirulence (Avr) genes to escape R-mediated plant immunity and identified a variety of mechanisms leading to virulence. The poplar rust fungus Melampsora larici-populina is the most damaging pathogen of poplars. A major adaptive event occurred in 1994 with the breakdown of RMlp7 resistance gene in poplar in Europe. Population genomics studies identified a locus in the genome of M. larici-populina, which likely corresponds to the AvrMlp7 candidate avirulence gene. We used a population genetics approach combined with dedicated qPCR assays on a comprehensive set of 281 isolates, covering 27 years (encompassing the resistance breakdown event), to validate the candidate locus and to assess its polymorphism. We found two mechanisms, a point mutation and a deletion, that allowed the pathogen to escape RMlp7-mediated resistance. Six diploid genotypes were thus characterized at the candidate locus (three avirulent and three virulent). In addition, a temporal analysis revealed that the two virulence alleles pre-existed (harbored as avirulent heterozygous genotypes) since the early samplings and were found in association (as virulent genotypes) at the time of the resistance breakdown. Our study documented that, in a diploid pathogen, combining virulence determinisms is adaptive.