Strong yellow color, caused by carotenoid accumulation, in semolina flour made from durum wheat (Triticum turgidum L. subsp. durum (Desf.)) is one of the most important traits for pasta production. The first step in the carotenoid biosynthesis pathway, which is catalyzed by phytoene synthase (PSY), is a bottleneck, and allelic variation of Psy-A1 in durum wheat produces different yellow pigment contents (YPC) in seeds. Durum wheat carrying leaf rust resistance gene Lr19, which was translocated from wheat relative Thinopyrum ponticum chromosome 7E to durum wheat chromosome 7A, is known to produce high YPC, and the causal gene is presumed to be Psy-E1, which is tightly linked to Lr19. In this study, Psy-E1 produced higher YPC than Psy-A1 alleles, such as Psy-A1k, Psy-A1l and Psy-A1o, in durum wheat. Segregation analysis demonstrated that Psy-E1 is located at the Psy-A1 locus on chromosome 7A. In a 2-year field test of near-isogenic materials, Psy-E1 was accompanied by yield loss with decreases in grain number per spike, test weight and thousand-kernel weight under moisture conditions typical of wheat-growing areas of Japan. Thus, Psy-E1 has the potential to contribute high YPC in durum wheat breeding programs, although the applicable cultivation environments are limited.