The characterization of crop cultivars (varieties) will come to depend increasingly on molecular characters in addition to traditional morphological and agronomic characters. Three cultivars of Kentucky bluegrass (Poa pratensis L.), developed by the Plant Breeding Station Hladké Životice (PBHŽ), were characterized using sequences and PCR-RFLP (Polymerase Chain Reaction-Restriction Fragment Length Polymorphism) patterns from the non-coding plastid trnL-F region (trnL intron, 549 bp, and trnL-trnF intergenic spacer [IGS], 344 to 364 bp). These characters could be readily and repeatably determined not only for mature plants, but also for seedlings (less than 12 weeks old), which are difficult to distinguish morphologically. The method is quick and sensitive. When restricted with a combination of BsaJ I and Bsm I, cultivar Slezanka has one major band, Moravanka has two, and Harmonie has three. When restricted with Alu I, the heaviest band migrates most slowly for Slezanka. It is expected that many Kentucky bluegrass cultivars will share the same trnL-F sequence, so these characters alone are not sufficient for variety identification.