The red flour beetle, Tribolium castaneum (Herbst) (Coleoptera: Tenebrionidae), is a major pest of stored grain and cereal crops. It is also an important model species in genetic, ecological, and evolutionary research. The majority of its genome was recently sequenced and published. However, the genomic sequence of the small Y-chromosome is still undetermined, which hinders the development of molecular sex identification methods. Traditional methods for sexing adult forms of Tribolium beetles are troublesome. Therefore, a method for molecular sex identification in the red flour beetle was developed. One sex-linked randomly amplified polymorphic DNA marker was converted into a sequence-characterized amplified region (SCAR). The SCAR was aligned with the T. castaneum reference whole-genome sequence and fully matched a fragment of a single contig of unknown genomic location. The novelty of the method is that the fragment consists of shorter DNA fragments that are also present at other locations around the genome, but the particular order of these fragments within the sequenced region appeared to be Y-specific and this property was utilized for marker development. A set of three primers for multiplex PCR reaction was designed resulting in amplification of different length Y-specific and not-Y-specific (control) DNA fragments in a single PCR, which allows to distinguish males from females. The primers successfully sexed pre-sexed pupae and adult beetles from six laboratory strains, showing that the order of the repeated fragments is conserved in the species and is not strain-specific.