As a member of the CLAVATA3 (CLV3)/EMBRYO SURROUNDING REGION (CLE) family, the dodecapeptide tracheary element differentiation inhibitory factor (TDIF) has a major impact on vascular development in plants. However, the influence of polymorphisms in the TDIF peptide motif on activity remains poorly understood. The model plant, provides a fast and effective tool for assaying the activity of homologs. Five homologs from a group of 93 genes in switchgrass (), a perennial biomass crop, named () genes were studied. The expression levels of , and were relatively high and all of them were expressed at the highest levels in the rachis of switchgrass. The precursor proteins for PvTDIFL1, PvTDIFL3, and PvTDIFL3 contained one, three, and two TDIFL motifs, respectively. Treatments with exogenous PvTDIFL peptides increased the number of stele cells in the hypocotyls of seedlings, with the exception of PvTDIFL_4p. Heterologous expression of in strongly inhibited plant growth, increased cell division in the vascular tissue of the hypocotyl, and disrupted the cellular organization of the hypocotyl. Although heterologous expression of and also affected plant growth and vascular development, PvTDIFL activity was not enhanced by the multiple TDIFL motifs encoded by and . These data indicate that in general, PvTDIFLs are functionally similar to TDIF but that the processing and activities of the PvTDIFL peptides are more complex.