A genetic linkage map was constructed in a backcross family of the red flour beetle, Tribolium castaneum, based largely on sequences from bacterial artificial chromosome (BAC) ends and untranslated regions from random cDNA's. In most cases, dimorphisms were detected using heteroduplex or single-strand conformational polymorphism analysis after specific PCR amplification. The map incorporates a total of 424 markers, including 190 BACs and 165 cDNA's, as well as 69 genes, transposon insertion sites, sequence-tagged sites, microsatellites, and amplified fragment-length polymorphisms. Mapped loci are distributed along 571 cM, spanning all 10 linkage groups at an average marker separation of 1.3 cM. This genetic map provides a framework for positional cloning and a scaffold for integration of the emerging physical map and genome sequence assembly. The map and corresponding sequences can be accessed through BeetleBase (http://www.bioinformatics.ksu.edu/BeetleBase/).