BLAST (Basic Local Alignment Search) analyses of Hordeum chilense RAPD markers have shown DNA sequence similarities with several plant genes. Biologically significant hits were scored for: plastocianin (Hordeum vulgare), alpha-gliadin (Triticum aestivum), Grandel-6 retrotransposon (Zea diploperennis), retrofit (gag/pol) copia-like, transposon-like element (Oryza longistaminata), copia-like retrotransposon Hopscotch polyprotein (gag/pol) (Zea mays) and several retrotransposons/retroelements from other species (Arabidopsis thaliana, Oryza sativa, Pisum sativum and Zea mays). Statistically significant hits also included endochitinase (Brassica napius), ribulose-1,5-bisphosphate carboxylase (Euglena gracilis), piruvate, orthophosphate dikinase (Flaveria trinervia), and an ORF flanked by ARS sequences (Triticum aestivum). Interestingly, it was also found that the currently recommended BLAST algorithm (version 2.0.11; gapped) at <http://www.ncbi.nlm.nih.gov> did not produce any biologically significant hits, whereas the previous version (1.4.11; ungapped) did generate biologically significant results. Thus, we suggest using the new BLAST 2.0.x when strong homologies are expected in short sequence stretches between closely related species. In other instances (and particularly when searching for lower similarities, yet spanning significantly larger stretches between more distant species), the BLAST 1.4.x could yield additional results. BLAST searches are valuable tools to identify and clone DNA sequences of interest, with applications in Marker-Assisted Selection (MAS) and wheat introgression.