Members of the YUCCA gene family encode a flavin monooxygenase-like enzyme that catalyzes the final step in auxin biosynthesis, the conversion of indole-3-pyruvic acid (IPyA) to indole-3-acetic acid (IAA). As this is generally considered the rate limiting step in IAA biosynthesis, the YUCCA genes are thought to be central to this process. In order to better understand the sites of IAA biosynthesis in shoot systems of hybrid poplar, we measured the expression levels of multiple YUCCA genes in the leaves of young, greenhouse-grown Populus tremula x alba clone INRA 717-1BA using qRT-PCR, normalizing expression against three reference genes. Previous work had shown that shoot apices (defined as tight clusters of young leaves at the apex with indistinguishable internodes) contained the highest concentrations of IAA, and that IAA dropped down to low and stable levels by L16, the 16th leaf beneath the apex. Both YUC1 and YUC12 followed a pattern similar to IAA concentrations, with peak expression in apices, but YUC12 showed more than 10x the expression of YUC1 and was consistently the dominant YUCCA expressed in whole leaves that were still undergoing expansion. In order to determine relative expression levels within leaves, L8 – which is about one-half fully expanded in poplars of this size and age – was further dissected into margin, blade, midvein and petiole. YUC2 and YUC12 were both highly expressed in the blade and leaf margin, but YUC2 dropped down to near-negligible levels in the midvein and petiole while YUC12 expression remained high. A neighbor-joining phylogenetic tree supports multiple pairs of YUCCA homologs in Populus, and suggests differential expression in leaf development within at least some of these pairs. These results provide important insight into the source(s) of leaf-derived IAA during both leaf expansion and cambial development in the stem.