The traditional raising system for Iberian pigs includes an open-air fattening period (montanera) where animals have a diet dominated by acorns. This system allows obtaining dry-cured products of unmatched quality. The most usual method for the verification of montanera products relies on assessing the particular backfat fatty acid composition after slaughter. Given that the advances on nutrition science makes feasible to emulate montanera meat products in an industrial farming environment, there is a growing need for the identification of complementary certification approaches. The objective of this study of TREASURE project was to evaluate the potential of gut microbiota composition analyses from faecal samples collected at slaughter as a new discrimination method of the montanera production system. Gut microbiota composition of 131 Iberian pigs (92 montanera and 131 commercial feeds) was determined by re-sequencing the bacterial 16S gene in an Illumina MiSeq device. Bioinformatic analyses were performed using Qiime’s subsampled open-reference OTU calling method. NMDS and PERMANOVA analyses performed using the Vegan R package showed significant effects of diet and sampling batch on gut microbiota composition. The microbiota of montanera-raised pigs showed a tendency towards a reduced microbial diversity. Differential abundance analyses performed with the metagenomeSeq R package confirmed these differences by identifying 997 out of 1,703 OTUs whose abundance was significantly different between both systems. Our results suggest that the gut microbiota composition of Iberian pigs sampled at the slaughter could be used as a supplementary certification tool of traditional montanera Iberian pigs. Funded by European Union’s H2020 RIA program (grant agreement 634476).