Although the Galápagos islands represent a unique ecosystem—with generally well-studied biota—research on its macroalgal flora remains relatively scant compared to other groups, particularly terrestrial fauna. Furthermore, while a few recent studies have applied molecular techniques, taxonomic identification of the archipelago’s macroalgae has mostly relied on morphology alone, which presents numerous challenges. In this study, we developed a DNA barcoding approach to identify macroalgal specimens collected opportunistically across the foraging grounds of endemic marine iguanas (Amblyrhynchus cristatus Bell 1825), which feed almost exclusively on macroalgae in coastal zones. We optimized primers for short DNA fragments of two genes and generated a reference dataset of macroalgal DNA sequences to facilitate future metabarcoding studies on marine iguana diet. Our approach—amplifying ~ 110 bp of the nuclear small subunit (18S) ribosomal gene—proved effective for identifying red, green, and brown macroalgae at higher taxonomic ranks, specifically at ordinal rank, while 184 bp of the ribulose-1,5-bisphosphate carboxy-lase/oxygenase (rbcL) gene enabled genus-rank identification in red macroalgae. We present the first compendium of macroalgal DNA sequences from marine iguana foraging areas, comprising 181 algal specimens: 136 red algae, 32 green algae, and 13 brown algae. We confirm taxa previously reported for the Galápagos and report eight new species of red macroalgae for this region. Our short barcodes provide a dataset of reference sequences to further research on marine iguana dietary habits. We also updated the Galápagos species checklists for Rhodophyta, Chlorophyta, and Ochrophyta by incorporating our newly generated molecular data alongside previously available macroalgal DNA sequences.
