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Peroxidase (POD) activity in wheat (Triticum aestivum L.) grain influences natural carotenoid pigment content and is associated with the color of flour, and processing and product quality. Here, we report the molecular characterization and physical mapping of POD genes in bread wheat. The complete genomic DNA (gDNA) sequence of two POD genes (TaPod-A2 and TaPod-D1), and the partial gDNA sequence of two additional POD genes (TaPod-A3 and TaPod-B1) from wheat were characterized using in silico cloning and validated through laboratory experiments. Using a set of 21 nullisomic-tetrasomic (NT) lines, six group-7 ditelosomic (Dt) lines, and 38 group-7 deletion (Del) lines of Chinese Spring (CS), TaPod-A2 and TaPod-D1 were found to be physically located on 0.73–0.83 and on the most distal 0.39 fraction arm length (FL) of 7AS and 7DS in cv. CS, respectively; whereas, TaPod-A3 and TaPod-B1 were assigned to the 0.40–0.49 and 0.40–0.48 FL of 7AL and 7BL, respectively. Based on single nucleotide polymorphisms (SNPs) of two alleles at the TaPod-D1 locus, two functional markers POD-7D1 and POD-7D6 were developed, amplifying 540- and 640-bp, fragments in varieties with higher and lower POD activities, respectively. A total of 224 wheat varieties were analyzed and showed a significant association between the polymorphic fragments and POD activity using POD-7D1 and POD-7D6 markers. The analysis of variance (ANOVA) indicated the average POD activities of 115 varieties with TaPod-D1a were significantly lower than 109 varieties with TaPod-D1b (P < 0.01). This study provides useful information of the POD genes in bread wheat, insight into wheat genome synteny and structure, gene-specific markers, and contributes a valuable resource for quality improvement in wheat breeding programs.