Nitrate transporters (NRTs) are regulators of nitrate assimilation and transport. The genome sequences of TaNRT2L12-A, -B and -D were cloned from wheat (Triticum aestivum L.), and polymorphisms were analyzed by sequencing. TaNRT2L12-D in a germplasm population was highly conserved. However, 38 single nucleotide polymorphisms (SNPs) in TaNRT2L12-A coding region and 11 SNPs in TaNRT2L12-B coding region were detected. Two derived cleaved amplified polymorphic sequences (dCAPS) markers A-CSNP1 and A-CSNP2 were developed for TaNRT2L12-A based on SNP-351 and SNP-729, and three haplotypes were identified in the germplasm population. B-CSNP1 and B-CSNP2 were developed for TaNRT2L12-B based on SNP-237 and SNP-1227, and three haplotypes were detected in the germplasm population. Association analyses between the markers and agronomic traits in 30 environments and phenotypic comparisons revealed that A-CSNP2-A is a superior allele of shorter plant height (PH), length of penultimate intode (LPI) and peduncle length (PL), B-CSNP2-G is a superior allele of higher grain number per spike (GNS). Hap-6B-1 containing both superior alleles B-CSNP1-C and B-CSNP2-A is a superior haplotype of 1000-grain weight (TGW). Expression analysis showed that TaNRT2L12-B is mainly expressed in the root base and regulated by nitrate. Therefore, TaNRT2L12 may be involved in nitrate transport and signaling to regulate TGW in wheat. The superior alleles and dCAPS markers of TaNRT2L12-A/B are beneficial to genetic improvement and germplasm enhancement with molecular markers-assisted selection.