Previous studies have identified two distinct O-methyltransferases (OMTs) implicated in isoflavonoid biosynthesis in Medicago species, a 7-OMT methylating the A-ring 7-hydroxyl of the isoflavone daidzein and a 4'-OMT methylating the B-ring 4'-hydroxyl of 2,7,4'-trihydroxyisoflavanone. Genes related to these OMTs from the model legume Medicago truncatula cluster as separate branches of the type I plant small molecule OMT family. To better understand the possible functions of these related OMTs in secondary metabolism in M. truncatula, seven of the OMTs were expressed in E. coli, purified, and their in vitro substrate preferences determined. Many of the enzymes display promiscuous activities, and some exhibit dual regio-specificity for the 4' and 7-hydroxyl moieties of the isoflavonoid nucleus. Protein structure homology modeling was used to help rationalize these catalytic activities. Transcripts encoding the different OMT genes exhibited differential tissue-specific and infection- or elicitor-induced expression, but not always in parallel with changes in expression of confirmed genes of the isoflavonoid pathway. The results are discussed in relation to the potential in vivo functions of these OMTs based on our current understanding of the phytochemistry of M. truncatula, and the difficulties associated with gene annotation in plant secondary metabolism.