Recommended rates of glyphosate for noncultivated areas destroy the aboveground shoots of the perennial plant leafy spurge. However, such applications cause little or no damage to underground adventitious buds (UABs), and thus the plant readily regenerates vegetatively. High concentrations of glyphosate, applied under controlled environmental conditions, have been shown to cause sublethal effects in UABs of leafy spurge that produce stunted and bushy phenotypes in subsequent generations of shoots. We treated leafy spurge plants in the field with glyphosate (0, 1.1, 3.4, or 6.7 kg ai ha−1) to determine its effects on vegetative growth from UABs and on molecular processes. The number of shoots derived from UABs of glyphosate-treated plants was significantly increased compared to controls in subsequent years after application, and new shoots displayed various phenotypical changes, such as stunted and bushy phenotypes. Quantifying the abundance of a selected set of transcripts in UABs of nontreated vs. treated plants (0 vs. 6.7 kg ha−1) indicated that glyphosate impacted molecular processes involved in biosynthesis or signaling of tryptophan or auxin (ARF4, CYP79B2, PIN3, TAA1, TRP6, YUC4), gibberellic acid (GA1/CPS1, GA2/KS), ethylene (ACO1, ACS10), cytokinins (AHP1, AK2, CKX1), and the cell cycle (CDC2A, CDC2B, CYCD3;1). Glyphosate-induced effects on vegetative growth and transcript abundance were persistent for at least 2 yr after treatment. Determining the molecular mechanisms associated with vegetative reproduction in leafy spurge following foliar glyphosate-treatment could identify limiting factors or new targets for manipulation of plant growth and development in perennial weeds.