Analysis of Y chromosome Y-STRs has proven to be a useful tool in the field of population genetics, especially in the case of closely related populations. We collected DNA samples from 169 males of Czech origin, 80 males of Slovakian origin, and 142 males dwelling Northern Poland. We performed Y-STR analysis of 12 loci in the samples collected (PowerPlex Y system from Promega) and compared the Y chromosome haplotype frequencies between the populations investigated. Also, we used Y-STR data available from the literature for comparison purposes. We observed significant differences between Y chromosome pools of Czechs and Slovaks compared to other Slavic and European populations. At the same time we were able to point to a specific group of Y-STR haplotypes belonging to an R1a haplogroup that seems to be shared by Slavic populations dwelling in Central Europe. The observed Y chromosome diversity may be explained by taking into consideration archeological and historical data regarding early Slav migrations. Am J Phys Anthropol 142:540–548, 2010. © 2010 Wiley-Liss, Inc.