Aluminum (Al) toxicity is a major stress factor limiting agricultural production on highly acidic soils. These types of soils, with pH values [LESS-THAN OR EQUAL TO] 5.0, are distributed over 70% of the world's potentially arable land. Rice (O. sativa L.) is one of the most Al tolerant species among smallgrain cereals, and within rice, cultivars from the varietal group Japonica are more tolerant than the Indica cultivar types. The genetic architecture of Al tolerance in rice is complex involving several genes and physiological mechanisms. To explore the natural genetic variation of Al tolerance in rice we developed 4 sets of reciprocal near isogenic lines (NILs), each targeting one of four loci associated with Al tolerance on chromosomes 1, 2, 9, and 12 from a cross between the Al susceptible indica cultivar, IR64, and the Al tolerant tropical japonica cultivar, Azucena. Phenotypic evaluation for Al tolerance in the NILs validated the effects of the QTLs on chromosomes 1, 9, and 12. We further explored the genetic basis of Al tolerance at the QTL on chromosome 12 by implementing a recombinant fine-mapping approach and narrowed down the target region to a ~44 Kb locus that contained 6 predicted genes, including ART1, a transcription factor known to regulate the expression of 31 genes in an Al-dependent manner, many of them involved in different Al tolerance mechanisms. These newly developed NILs, and two sets of interspecific introgression lines (ILs), constructed using two wild accessions as donors, O. rufipogon Griff., and O. meridionalis Ng., and a common recurrent parent, cv. Curinga (O. sativa ssp. tropical japonica), represent valuable germplasm resources for understanding the genetic basis of Al tolerance in rice. They can be used as parental materials for fine mapping and positional cloning efforts and as the basis for detailed molecular physiology studies. This work is also the first effort to confirm whether ART1, our strongest candidate gene in the 44 Kb region, is responsible for the Al tolerance effect of the QTL on chromosome 12.