Plant peroxidases are a family of related proteins possessing highly conserved domains. Degenerate oligonucleotide primers based on these conserved domains can be used to amplify DNA sequences coding for peroxidases from plants with unsequenced genomes. Polymorphisms in peroxidase genes among buffalograss [Buchloe dactyloides (Nutt.) Engelm.] genotypes and eight other grasses were evaluated, and potential evolutionary relationships were deduced using this approach. Fourteen peroxidase specific primers with alternative forward and reverse primers using 34 rice peroxidase cDNAs were designed based on conserved motifs of this gene family. Targeted polymerase chain reaction (PCR) amplification of genomic DNA from 28 buffalograss, 4 C4, and 4 C3 grass genotypes yielded polymorphisms, differentiating diploids from polyploids within buffalograss and C3 and C4 grass species from each other. A total of 11 peroxidase gene fragments, 7 belonging to buffalograss and 4 to the other grass species, were sequenced. Five of these sequences were clustered with rice (Oryza sativa L.) ascorbate peroxidase known to have chloroplast origin. These results demonstrate that primers targeting the peroxidase gene family can be used to study genotypic diversity and evolutionary relationships on an intraspecific and interspecific basis. The PCR-based peroxidase markers may also have potential for linkage mapping and differential gene expression studies in grasses.