In this work, eight human-specific Alu insertion polymorphisms (ACE, TPA25, PV92, APO, FXIIIB, D1, A25, and B65) were typed in 106 unrelated healthy individuals born in the province of Valencia (Spain), with the aim of analyzing the genetic relationships between this region of the Iberian Peninsula and other Mediterranean populations. To that end, Alu data on Eastern European, Western European, and North African populations were compiled from previous studies. The genetic information was stressed by means of genetic distances (R matrix method), nonmetric multidimensional scaling (MDS) and analyses of molecular variance (AMOVA). In Valencia, the most common Alu insertion was APO (0.940), and the least frequent was A25 (0.104). The average gene diversity (GD) computed for the sample examined was comparatively high (0.382). The insertion frequencies estimated for the eight Alu markers were very similar to the mean frequencies calculated for the whole set of populations included in the study, suggesting the hybrid nature of the Valencia's gene pool. MDS and AMOVA results generated from Alu data reveal that the Mediterranean has acted as a strong genetic boundary between the north (Europe) and the south (Northern Africa), resulting in significant gene diversity between the populations of the two regions. Restricted exclusively to the European scope, we suggest the possibility that the Mediterranean could have also acted as a migratory passageway, propitiating the dissemination of cultures and genes between the east and west of Europe and giving rise to some homogenization of gene frequencies among coastal dwelling populations. Am. J. Hum. Biol. 18:187-195, 2006. (c) 2006 Wiley-Liss, Inc.