The yellow pigment (YP) of durum wheat (Triticum turgidum L. var durum) semolina is due in part to the presence of carotenoid pigments found in the endosperm and is an important end-use quality trait. Phytoene synthase (Psy) is considered a rate-limiting enzyme in the carotenoid biosynthetic pathway and in this study, three alleles of Psy1-A1 were sequenced from four durum wheat cultivars and a co-dominant marker was developed for genetic mapping. Psy1-A1 mapped to chromosome 7AL near Xwmc809 in three durum mapping populations and was significantly associated with a pigment quantitative trait loci (QTL) identified on that chromosome. A second QTL localized 25 cM proximal to Psy1-A1 in two populations, and the interaction between the two QTL was not significant. Consistent with QTL mapping data, the Psy1-A1o allele was associated with elevated pigment in a validation population comprising 93 diverse cultivars and breeding lines. These results confirm an earlier hypothesis that Psy1, and at least one additional gene in the distal region of 7AL, are associated with grain YP differences in durum wheat. The functional co-dominant marker developed in this study differentiates the Psy1-A1 alleles reported here and could be used as a target to enhance YP selection in durum wheat breeding programs.