Aluminum (Al) toxicity is the major constraint for crop productivity in acid soils. Wild rye species (Secale spp.) exhibited high Al-tolerance being a good source of genes related to this trait. The Alt1 locus located on 6RS chromosome arm is one of the four main loci controlling Al tolerance in rye and is known to harbor major genes but, so far, none has been found. Through the synteny among the short arm of the rye chromosome 6R and the main grass species, we found a candidate MATE gene for the Atl1 locus, posteriorly named ScMATE3, which was isolated and characterized in different Secale species. The sequence comparisons revealed both intraspecific and interspecific variability with high sequence conservation in the Secale genus. SNPs with replacement substitution that changed the structure of the protein and can be involved in the Al tolerance trait were found in ScMATE3 gene. The predicted subcellular localization of ScMATE3 is the vacuolar membrane which together with the phylogenetic relationships performed with other MATE genes of the Poaceae family related to Al detoxification, suggest involvement of ScMATE3 in an internal tolerance mechanism. Moreover, expression studies of this gene in rye corroborate his contribution in some Al resistance mechanism. The ScMATE3 gene is located on 6RS chromosome arm between the same markers that Alt1 locus involved in Al resistance mechanisms in rye being a good candidate gene.