Over the course of its long history, Armenia has acted as both a source of numerous indigenous cultures and as a recipient of foreign invasions. As a result of this complex history among populations, the gene pool of the Armenian population may contain traces of historically well-documented ancient migrations. Furthermore, the regions within the historical boundaries of Armenia possess unique demographic histories, having hosted both autochthonous and specific exogenous genetic influences. In the present study, we analyze the Armenian population sub-structure utilizing 17 Y-chromosome short tandem repeat (Y-STR) loci of 412 Armenians from four geographically and anthropologically well-defined groups (Ararat Valley, Gardman, Lake Van and Sasun). To place the genetic composition of Armenia in a regional and historic context, we have compared the Y-STR profiles from these four Armenian collections to 18 current-day Eurasian populations and two ancient DNA collections. Our results illustrate regional trends in Armenian paternal lineages and locale-specific patterns of affinities with neighboring regions. Additionally, we observe a phylogenetic relationship between the Northern Caucasus and the group from Sasun, which offers an explanation for the genetic divergence of this group from other three Armenian collections. These findings highlight the importance of analyzing both general populations as well as geographically defined sub-populations when utilizing Y-STRs for forensic analyses and population genetics studies.