Colletotrichum fructicola is the main species causing apple bitter rot (ABR) and Glomerella leaf spot (GLS) in southern Brazil, and ABR in Uruguay where GLS remains unnoticed. Thus, this work aimed to determine the genetic structure of C. fructicola isolates of both the countries. A total of 28 out of 31 Brazilian isolates (90.3%) caused typical symptoms of GLS, while only 6 of 25 Uruguayan isolates (24.0%) originating from fruits were able to infect leaves, but causing atypical symptoms. Both populations showed similar levels of Nei's gene diversity (h = 0.088 and 0.079, for Brazilian and Uruguayan populations, respectively), and Bayesian cluster analysis inferred two genetic clusters correlated with the geographical origin of isolates. A principal coordinates analysis scatter plot and an unweighted pair group method with arithmetic mean-based dendrogram also grouped Brazilian and Uruguayan isolates into two groups. By pairwise comparison of nitrate-nonutilizing (nit) mutants with a proposed set of testers, all Uruguayan isolates were grouped into a unique vegetative compatibility group (namely VCG 1), while Brazilian isolates were grouped into four VCGs (VCG 1 to 4). Brazilian and Uruguayan populations of C. fructicola were found to be genetically distinct. Our results suggest that isolates of C. fructicola from Brazil capable of causing GLS and ABR arose independently of those from Uruguay. Possible causes leading to the evolutionary differences between populations are discussed.