Arachnids are the most abundant land predators. Despite the importance of their functional roles as predators and the of necessity to understand their diet for conservation and nutrient fluxes, the trophic ecology of many arachnid species is not fully understood. In the case of the wandering spider, Phoneutria boliviensis F. O. Pickard-Cambridge, 1897, only selected field and laboratory observational studies about their diet exist. By using a DNA metabarcoding approach, we compared the prey found in the gut content of males and females from three distant Colombian populations of P. boliviensis. By DNA metabarcoding of the cytochrome c oxidase subunit I (COI), we detected and identified 234 prey records belonging to 96 operational taxonomic units (OTUs), as prey for this wandering predator. Our results broaden the known diet of P. boliviensis with at least 75 prey taxa not previously registered in fieldwork or laboratory experimental trials. These results suggest that P. boliviensis feeds predominantly on invertebrates (Diptera, Lepidoptera, Coleoptera and Orthoptera) and opportunistically on small squamates. Intersex and interpopulation differences are observed. Assuming that prey preference does not vary between populations, these differences are likely associated with a higher local prey availability. Finally, we suggest that DNA metabarcoding can be used for evaluating subtle differences in the diet of distinct populations of P. boliviensis, particularly when predation records in the field cannot be established or quantified using direct observation