High-density Single Nucleotide Polymorphism (SNP) scans of the human genome have been applied in many populations worldwide to investigate their genetic characteristics. However, populations in North Africa, an isolated subcontinental area between Sub-Sahara Africa and Europe, have not been examined. In the present study, seven North African populations and four neighboring Spanish populations are analyzed using a high-density SNP microarray. North African populations appear to form a clinal pattern of genetic differentiation between Sub-Saharan Africans and Europeans, being much more similar to Europeans than to Sub-Saharans. The genetic similarity between North African populations exhibits an east-west gradient pattern corresponding to their geographic locations. High and varying levels of autozygosity, as well as a potentially indigenous genetic component, are observed in North Africans. Noticeably, Tunisians turn out to be the North African population most distinct from Europeans and Sub-Saharans, and have the highest levels of autozygosity. Basques can be clearly distinguished from other Spanish populations, as being more similar to the Western Europeans, and also have the largest number of fixed ancestral and derived alleles among all the populations studied. The ancestral allele frequency distribution of Basques is most similar to that of East Asians, suggesting a small effective population size. All these results indicate that the Basque population is a genetic isolate distinct from the surrounding Spanish populations as well as from other Southern and Western European populations, although the magnitude of genetic differentiation is subtle.