P23k is a 23 kDa protein involved in sugar translocation in the scutellum of germinating barley seeds. The present study was carried out to provide the genomic characterization for P23k gene in terms of copy number, chromosome mapping, genetic mapping and expression analysis in germinating sculletum in two major Triticeae crops, barley and wheat, and their relatives. Southern blotting showed that a variable copy number with different restriction fragment sizes was found among 15 Hordeum accessions, while low copy number were found to be conserved in 23 Triticum and 3 Aegilops accessions. Genetic and physical mapping study identified that the P23k gene is duplicated in wild and cultivated barley on chromosomes 1H, 2H, and 3H, and further tandem duplication on chromosomes 1H and 3H. In contrast, the wheat P23k is located on chromosome 3A of durum wheat and at the distal portion of the long arms of 3A and 3D chromosomes of bread wheat. Northern blotting showed remarkably high accumulation of P23k transcript in the germinating scutellum in cultivated and wild barley, whereas very few or no accumulation was detected in diploid, tetraploid, and hexaploid wheat accessions. The present study suggests a simple scenario where the ancestral P23k is encoded on the distal portion of an ancestral chromosome of homoeologous chromosome 3. Beside of polyploidy, dispersed and tandem duplications could trigger generation of the P23k family in the Hordeum lineage, while an ancestral P23k has been conserved in homoeologous 3A and 3D chromosomes in the wheat lineage.