Abstract The Guatemala Suture Zone (GSZ), Guatemala, is a region that contains two distinct suture-related serpentinite mélanges straddling the Motagua fault and an ophiolitic complex paired with the northern mélange. The serpentinite matrix of the mélanges formed by subduction-fluid hydration of peridotite from the deep mantle wedge. The occurrence of serpentinite from both exhumed subduction channel mélange and ophiolite is not uncommon in paleo–suture zones, but distinguishing them and their tectonic origin can be difficult. A new method of discrimination, based on boron isotopes in serpentine from both mélanges and ophiolite, as well as on mica and pyroxene from the metamorphic and vein-rock blocks embedded within the mélanges, has been developed. The metamorphic and vein samples have mainly negative δ11B, ranging from –15.3‰ to +4.3‰, in the same range as the serpentine from the mélanges (–14.4‰ to +9.7‰). In addition to being the most negative δ11B values ever measured in serpentinite, comparable values from vein minerals indicate that the same fluid serpentinized the overlying mantle. In contrast, serpentine samples from the ophiolite have positive δ11B, in the range 0‰ to +18.0‰, consistent with hydration by seawater-derived fluids. As the GSZ displays two mélanges whose serpentinite originated from two different deep subductions and mantle hydration, we hypothesize that the negative signature of exhumed mélange serpentine is the norm and that the B isotopic signature can be a useful tool to discriminate the tectonic origin of serpentinization in paleo–suture zones.