The δ13C value of fossil plant materials can be used to gain insight into the dominant photosynthetic pathway, as well as other environmental attributes, of ancient plant ecosystems. Nucleotide sequences from land plant nucleic acids extracted from 400 ka fossil sediments have been recognized as the oldest authenticated fossil deoxyribonucleic acid (DNA), making the inference of plant taxonomy possible in substrates devoid of plant macrofossils and microfossils. If the C isotope relationship between bulk plant tissue and associated plant nucleic acids were known, fossil plant nucleic acids could be analyzed for δ13C value and used as land plant isotopic substrates within mixed organic material. Toward this end, we present δ13C analyses of nucleic acids isolated from 12 higher plant species that span the full phylogenetic diversity of seed plants. Extracted nucleic acids were dominated by double-stranded DNA containing fragments of rbcL gene ∼350 base pairs in length. The C isotope compositions of plant nucleic acids were found to be enriched in 13C relative to bulk plant tissue by a constant value = 1.39‰. This study represents the first comparison of the δ13C value of nucleic acids to the δ13C value of bulk tissue for multicellular organisms; our results contrasted with the minimal fractionations reported for microorganisms. Because the isotopic enrichment is constant across tracheophytes, the δ13C value of fossil plant DNA can be used as a paleoenvironmental indicator, eliminating the need for morphological recognition of fossil plant material in paleoenvironmental studies.