Phakopsora pachyrhizi, the fungal pathogen that causes Asian soybean rust, has the potential to cause significant losses in soybean yield in many production regions of the United States. Germplasm with durable, single-gene resistance is lacking, and control of rust depends on timely application of fungicides. To assist the development of new modes of soybean resistance, we identified peptides from combinatorial phage-display peptide libraries that inhibit germ tube growth from urediniospores of P. pachyrhizi. Two peptides, Sp2 and Sp39, were identified that inhibit germ tube development when displayed as fusions with the coat protein of M13 phage or as fusions with maize cytokinin oxidase/dehydrogenase (ZmCKX1). In either display format, the inhibitory effect of the peptides on germ tube growth was concentration dependent. In addition, when peptides Sp2 or Sp39 in either format were mixed with urediniospores and inoculated to soybean leaves with an 8-h wetness period, rust lesion development was reduced. Peptides Sp2 and Sp39, displayed on ZmCKX1, were found to interact with a 20-kDa protein derived from germinated urediniospores. Incorporating peptides that inhibit pathogen development and pathogenesis into breeding programs may contribute to the development of soybean cultivars with improved, durable rust tolerance.