Two human leukocyte antigen (HLA) class I loci (HLA-A and HLA-B) and one class II locus (HLA-DR) were typed at the DNA level in the Sicilian population. Study participants were of Sicilian origin (183 for class I loci and 260 for class II loci) and live in three towns, chosen on the basis of geographic position and different historical events. These towns are Sciacca (southwest Sicily, located at sea level, conquered by Arabs in a.d. 814), Piana degli Albanesi (northwest Sicily, 720 m above sea level, has maintained religious, cultural, and linguistic peculiarities traced to Albanian settlement in 1488), and Troina (northeast Sicily, 1,120 m above sea level, known as the first settlement of Normans). The assumptions underlying the study of genetic structure, based on HLA allele polymorphism, are that these three towns are located in areas that can be distinguished according to historical criteria and that they are likely to have contributed to cultural and probably genetic differences. As such, the high frequency of some alleles in Sciacca and Troina seems to be correlated with Greek, Phoenician, North African, and Arab influence. In accordance with different human settlements in Sicily, we found that the HLA allele frequencies support the existence of genetic differentiation between the western and eastern sides of Sicily. This separation is attributed to Greek colonization in the east and to Phoenician-Carthaginian-Arab influence in the west. Moreover, the comparisons of all allele frequencies between Mediterranean and African populations show the same trend, highlighting in some cases European origin and in other cases non-European origin.