Rye is a diploid crop species with many outstanding qualities, and is important as a source of new traits for wheat and triticale improvement. Rye is highly tolerant of aluminum (Al) toxicity, and possesses a complex structure at the Alt4 Al tolerance locus not found at the corresponding locus in wheat. Here we describe a BAC library of rye cv. Blanco, representing a valuable resource for rye molecular genetic studies, and assess the library's suitability for investigating Al tolerance genes. The library provides 6 x genome coverage of the 8.1 Gb rye genome, has an average insert size of 131 kb, and contains only ~2% of empty or organelle-derived clones. Genetic analysis attributed the Al tolerance of Blanco to the Alt4 locus on the short arm of chromosome 7R, and revealed the presence of multiple allelic variants (haplotypes) of the Alt4 locus in the BAC library. BAC clones containing ALMT1 gene clusters from several Alt4 haplotypes were identified, and will provide useful starting points for exploring the basis for the structural variability and functional specialization of ALMT1 genes at this locus.