Background Poa pratensis is one of the most common species of meadow grass in Europe. Most cultivars of the species found in Poland were originally derived from its ecotypes. We compared the effectiveness of the RAPD and ISSR methods in assessing the genetic diversity of the selected populations of P. pratensis. We examined whether these methods could be useful for detecting a possible link between the geographical origin of a given population and its assessed genetic variation. Methods The molecular markers RAPD and ISSR were used and their efficiency compared using, inter alia, statistical multivariate methods (UPGMA and PCA). Results The low value of Dice’s coefficient (0.369) along with the significantly high percentage of polymorphic products indicates a substantial degree of genetic diversity among the studied populations. Our results found a correlation between the geographical origin of the studied populations and their genetic variations. For ISSR, which proved to be the more effective method in that respect, we selected primers with the greatest differentiating powers correlating to geographical origin. Discussion The populations evaluated in this study were characterized by a high genetic diversity. This seems to confirm the hypothesis that ecotypes of P. pratensis originating from different regions of Central Europe with different terrain structures and habitat conditions can be a source of great genetic variability.