Low-level DNA N6-methyldeoxyadenosine (DNA-m6A) has recently been reported across various eukaryotes. Although anti-m6A antibody–based approaches are commonly used to measure DNA-m6A levels, this approach is known to be confounded by DNA secondary structures, RNA contamination, and bacterial contamination. To evaluate for these confounding features, we introduce an approach for systematically validating the selectivity of antibody-based DNA-m6A methods and use a highly selective anti-DNA-m6A antibody to reexamine patterns of DNA-m6A in C. reinhardtii, A. thaliana, and D. melanogaster. Our findings raise caution about the use of antibody-based methods for endogenous m6A quantification and mapping in eukaryotes.