Abscisic acid (ABA) plays a vital role in mediating abiotic stress responses in plants. De novo ABA biosynthesis involves cleavage of carotenoid precursors by 9-cis-epoxycarotenoid dioxygenase (NCED), which is rate controlling in leaves and roots; however, additional bottlenecks in roots must be overcome, such as biosynthesis of upstream carotenoid precursors. Phytoene synthase (PSY) mediates the first committed step in carotenoid biosynthesis; with PSY3 described here, maize (Zea mays) and other members of the Poaceae have three paralogous genes, in contrast to only one in Arabidopsis thaliana. PSY gene duplication has led to subfunctionalization, with each paralog exhibiting differential gene expression. We showed that PSY3 encodes a functional enzyme for which maize transcript levels are regulated in response to abiotic stresses, drought, salt, and ABA. Drought-stressed roots showed elevated PSY3 transcripts and ABA, responses reversed by rehydration. By blocking root carotenoid biosynthesis with the maize y9 mutation, we demonstrated that PSY3 mRNA elevation correlates with carotenoid accumulation and that blocking carotenoid biosynthesis interferes with stress-induced ABA accumulation. In parallel, we observed elevated NCED transcripts and showed that, in contrast to dicots, root zeaxanthin epoxidase transcripts were unchanged. PSY3 was the only paralog for which transcripts were induced in roots and abiotic stress also affected leaf PSY2 transcript levels; PSY1 mRNA was not elevated in any tissues tested. Our results suggest that PSY3 expression influences root carotenogenesis and defines a potential bottleneck upstream of NCED; further examination of PSY3 in the grasses is of value for better understanding root-specific stress responses that impact plant yield.