HMGB1 and HMGB2 are DNA-interacting proteins but can also have extracellular actions during inflammation. Despite their relatively high homology, they may have distinct roles, making it essential to be able to differentiate between the two. Here we examine the specificity of five commercially-available anti-HMGB1 antibodies. By Western blotting of recombinant proteins and HMGB1-/- mouse embryonic fibroblasts, we identified only one HMGB1 antibody that, under our experimental conditions, did not also detect HMGB2. Selecting specific antibodies for HMGB1 and HMGB2 allowed identification of distinct HMGB1 and HMGB2 subcellular pools in primary neutrophils.