Fusarium proliferatum (Matsushima) Nirenberg is one of the most important Fusarium species considering its worldwide distribution, its pathogenic ability in a wide range of crops, and its mycotoxigenic potential. F. proliferatum conidia are easily carried by rainwater and, in this work, five isolates from rainwater were evaluated as inoculum sources to produce garlic rot and their pathogenic ability were compared with six F. proliferatum isolates from diseased garlic. A phylogenetic analysis was also performed to unravel possible genetic differences among the isolates. Pathogenicity tests were performed in three garlic varieties (purple, white and purple Chinese) and the results revealed that the isolates from rainwater were pathogenic to each of them. However, in all cases, their pathogenic ability was lower than in the case of isolates obtained from rooted garlic. The phylogenetic analysis based on parsimony carried out with the hypervariable region of the intergenic transcribed spacer (IGS) in F. proliferatum showed genetic differences depending on the origin of the isolates. Isolates from rainwater were definitely separated in an independent branch from garlic isolates. This paper shows an interesting approach to the ecological significance of F. proliferatum as a fungal pathogen on garlic. Some degree of relation between genetic variability and pathogenicity was also demonstrated.