Human population movements in North Africa have been mostly restricted to an east-west direction due to the geographical barriers imposed by the Sahara Desert and the Mediterranean Sea. Although these barriers have not completely impeded human migrations, genetic studies have shown that an east-west genetic gradient exists. However, the lack of genetic information of certain geographical areas and the focus of some studies in parts of the North African landscape have limited the global view of the genetic pool of North African populations. To provide a global view of the North African genetic landscape and population structure, we have analyzed ∼2,300 North African mitochondrial DNA lineages (including 269 new sequences from Libya, in the first mtDNA study of the general Libyan population). Our results show a clinal distribution of certain haplogroups, some of them more frequent in Western (H, HV0, L1b, L3b, U6) or Eastern populations (L0a, R0a, N1b, I, J) that might be the result of human migrations from the Middle East, sub-Saharan Africa, and Europe. Despite this clinal pattern, a genetic discontinuity is found in the Libyan/Egyptian border, suggesting a differential gene flow in the Nile River Valley. Finally, frequency of the post-LGM subclades H1 and H3 is predominant in Libya within the H sequences, highlighting the magnitude of the LGM expansion in North Africa. Am J Phys Anthropol, 2011. © 2011 Wiley-Liss, Inc.