There has been increasing interest in recent years in sunn hemp (Crotalaria juncea), as a leguminous cover crop and green manure, for weed and pest management and improving soil health. Aqueous extracts and ground shoot tissue have previously been demonstrated to be phytotoxic. To further explore its allelopathic potential, bioassays and chemical characterization of water-soluble eluates of sunn hemp were undertaken. Lettuce (Lactuca sativa) radicle growth inhibition was conducted with aqueous eluates from thinly sliced sunn hemp leaves, stems, and seeds, and all three tissues exhibited the inhibitory potential. Fourteen accessions originating from the United States, India, Brazil, South Africa, Pakistan, and Nigeria had water-soluble allelochemicals in leaves, suggesting that allelopathic potential is widely distributed in this species. The highest level of inhibitory potential was found in accession IN-86. Further characterization of IN-86 leaf eluates indicated that the inhibitory compound(s) was/were not soluble in chloroform, but was/were stable when boiled for 15 minutes and resistant to 1 n HCl. Binding and elution from AG-1(OH−) ion-exchange resin also were observed. An analysis of leaf eluates of IN-86 using high-performance liquid chromatography (HPLC) followed by mass spectrometry (MS) showed the presence of a compound with a mass-to-charge ratio of 148, consistent with the spectrum for hydroxynorleucine, a phytotoxic nonprotein amino acid previously reported in seeds of C. juncea. However, its low concentration (<1 μg·mL−1) suggested that other components of the eluate were responsible for the observed allelopathic effect. The results indicate the feasibility for development of weed control strategies using allelochemicals derived from sunn hemp biomass of select genotypes IN-86, NG-71, and BR-20 from India, Nigeria, and Brazil, respectively.