Zeta-carotene desaturase (ZDS) is a key enzyme for carotenoid biosynthesis, demonstrating high association with the yellow pigment (YP) content in wheat grain. Cloning ZDS gene and developing functional markers are important for marker-assisted selection in wheat breeding. In the present study, the full-length DNA sequence of a ZDS gene on wheat chromosome 2A, designated TaZds-A1, was cloned, with 14 exons and 13 introns, and it has an open reading frame (ORF) of 1 707 bp, encoding 568 amino acid residues. A co-dominant functional marker, YP2A-1, was designed based on the polymorphisms of two alleles at the locus, TaZds-A1a and TaZds-A1b, yielding 183- and 179-bp fragments in TaZds-A1a and TaZds-A1b genotypes, respectively. A new QTL for YP content was detected on chromosome 2A, co-segregating with the functional marker YP2A-1 and TaZds-A1; it explained 11.3% of the phenotypic variance for YP content in a doubled haploid (DH) population from Zhongyou 9507/CA9632. Among 217 Chinese wheat cultivars and advanced lines, the average grain YP content of 126 cultivars with TaZds-A1b allele was 7.8% higher than that of 91 cultivars with TaZds-A1a allele.