Melampsora larici-populina Kleb. is a Eurasian species that causes leaf rust of many species of Populus from sections Tacamahaca and Aigeiros. This rust has been inadvertently introduced to many parts around the world affecting poplar species that grow naturally, which may be susceptible to this fungus. In Chile, early European settlers introduced poplars; rust, attributed to M. larici-populina, has been reported in these trees since 1918. However, a modern confirmation has been lacking, and pathogenic variation of the Chilean population of M. larici-populina has not been investigated. Using a morphological analysis of urediniospores and sequencing of internal transcribed spacer (ITS) regions, we confirmed the presence of M. larici-populina in Chile. ITS regions exhibited 100% homology with M. larici-populina. Scanning electron microscopy showed that spores were 26 to 47 µm in length, 13 to 16 µm in width, and echinulate except for apices, which are smooth, characteristics described for this species of rust. The variability of M. larici-populina is characterized by the presence of pathotypes, which allows the fungus to infect despite the resistance of certain poplar hybrids. We concluded that the identified spores belong to M. larici-populina, with virulences 1, 2, 3, 4, 5, and 6. These results describe variation in virulence of M. larici-populina, which suggests the presence of a sexual stage in Chile.