The regions of the Caucasus, Anatolia, and the Near East represent an extremely important area with respect to ancient population migration and expansion, and the spread of the Caucasian, Indo-European, Afro-Asiatic, and Altaic languages. We examined genetic variation within and between 12 ethno-territorial populations belonging to four major language families by using six microsatellites, or short tandem repeats (STR) and 12 Unique Event Polymorphism (UEP) loci mapped to the non-recombining portion of the human Y-chromosome. The applied set of markers did not unconditionally support the language replacement hypothesis for the populations under consideration. Moreover, regarding the South Caucasus and Anatolia, our results showed greater genetic distances between the populations from different language families, and close genetic affinity for the populations from the same language group—in this case, for the Turkophone ethnic units. The results point to the importance of appropriate Y-chromosome sampling procedures in geographically structured populations and to the necessity of using a relevant set of markers that provides best discrimination of populations with different genetic histories.