Technical Abstract: Phomopsis seed decay (PSD) of soybean is caused primarily by the fungal pathogen Phomopsis longicolla. PSD impairs seed germination, reduces seedling vigor, and can substantially reduce stand establishment. In hot and humid conditions, PSD can cause significant yield losses. Few studies have explored molecular mechanisms underlying virulence in P. longicolla. A major hindrance to functional genomics in P. longicolla has been the lack of a reliable genetic transformation system for the pathogen. As a foundation for future functional genomic research in the soybean pathogen P. longicolla, we developed a robust and reliable Agrobacterium tumefaciens-mediated transformation system. Transformants of P. longicolla were analyzed to assess the expression of the green florescent protein (GFP) and confirmed by Southern blot analysis.This is the first report of P. longicolla transformation, and will contribute a useful tool for insertional mutagenesis studies in an important pathogen of soybean.