Three populations from northern Pakistan, the Burusho, Kalash, and Pathan, claim descent from soldiers left behind by Alexander the Great after his invasion of the Indo-Pak subcontinent. In order to investigate their genetic relationships, we analyzed nine Alu insertion polymorphisms and 113 autosomal microsatellites in the extant Pakistani and Greek populations. Principal component, phylogenetic, and structure analyses show that the Kalash are genetically distinct, and that the Burusho and Pathan populations are genetically close to each other and the Greek population. Admixture estimates suggest a small Greek contribution to the genetic pool of the Burusho and Pathan and demonstrate that these two northern Pakistani populations share a common Indo-European gene pool that probably predates Alexander's invasion. The genetically isolated Kalash population may represent the genetic pool of ancestral Eurasian populations of Central Asia or early Indo-European nomadic pastoral tribes that became sequestered in the valleys of the Hindu Kush Mountains.