Main conclusion <p>Natural variation in <i>SlAMT1.1</i> among wild tomatoes may influence post-translational regulation, allowing sustained ammonium uptake under high N supply and providing alleles that can be employed to improve N uptake efficiency in crops.</p> Abstract <p>Cultivated plants, particularly tomato (<i>Solanum lycopersicum</i>), require substantial nitrogen (N) inputs to achieve high commercial yields. This demand often leads to the excessive application of costly N-based fertilizers during cultivation. Wild tomato species represent valuable genetic resources for enhancing N uptake efficiency. In many plants, ammonium is the preferred N source, transported by proteins of the AMMONIUM TRANSPORTERS (AMT) family. Here, we characterized the extensive genetic diversity of an <i>AMT1.1</i> ortholog across both cultivated and distantly related wild tomato species. Phylogenetic and diversity analyses revealed marked divergence in the <i>SlAMT1.1</i> sequence between cultivated tomato accessions and wild <i>Solanum</i> (section Lycopersicon) species. Comparative analyses of <i>SlAMT1.1</i> alleles from the Arcanum and Neolycopersicon groups showed enhanced uptake of ¹⁵N-labeled ammonium in roots under repressive ammonium resupply conditions. Notably, we found that the feedback inhibition of ammonium uptake, typical in domesticated tomato roots, was lost in these wild accessions, indicating the presence of a novel regulatory mechanism that adjusts uptake capacity across a wide range of ammonium availability. Our findings indicate that variation in the <i>SlAMT1.1</i> gene largely explains the observed differences in ammonium uptake between domesticated tomatoes and their wild relatives. Therefore, the natural genetic variation present in the wild tomato <i>SlAMT1.1</i> alleles offers valuable potential for genomic-based breeding strategies to sustainably improve ammonium uptake in crops.</p>