The chemical basis for wheat seedling allelopathy on the growth of annual ryegrass was investigated by the identification and quantification of multiple allelochemicals from wheat seedlings. Results indicated that 58 wheat accessions differed significantly in seedling allelopathy and inhibited the root growth of ryegrass from 10 to 91%, depending on accession. Analysis of allelochemicals by GC/MS/MS indicated that allelopathy was significantly correlated with the levels of measured allelochemicals in the shoots and roots of young wheat seedlings.