Soybean [Glycine max (L.) Merr.] is an important source of isoflavones used by the nutraceutical industry. The soybean genome (2n = 40, 975 Mb) has recently been sequenced, and over a million (redundant) gene tags (expressed sequence tags, ESTs) are available in public databases. Using bioinformatics, we investigated five key enzymes of the isoflavonoid pathway (i.e., chalcone isomerase, isoflavone synthase, 2-hydroxyisoflavanone dehydratase, isoflavanone-7-O-glycosyltransferase, and isoflavone-7-O-glucoside-6′′-O-malonyltransferase) to gain a better understanding of which gene homologues are expressed. Contiguous sequences (contigs) were assembled from EST data to represent the specific genes and were subsequently used to predict and verify known and novel gene homologues in the recently released chromosome-based assembly of the soybean genome. Novel transcripts for 2-hydroxyisoflavanone dehydratase and isoflavone-7-O-glucosyltransferase were discovered in these data and in silico expression profiles are presented for all the genes identified in the isoflavonoid pathway. Key words: Soybean, expressed sequence tag, isoflavonoid, gene expression, homologues