The aim of this study was to select the best arrangements of MP–PCR (microsatellite-primed PCR) for routine large-scale fingerprinting of flax cultivars. We found optimum PCR conditions for the application of five previously published primers (PCT1–PCT5) to flax cultivar fingerprinting. We modified to optimum MP–PCR which was targeted to flax tetrameric [GATA] microsatellite loci specified by primer PCT6. We found that after a reamplification PCR step was involved we were able to generate highly discriminating fingerprinting patterns, which distinguished all eight flax cultivars individually. In particular primers 3PCT1 and 3PCT2 were promising for future large-scale fingerprinting due to the production of most polymorphic bands. Increasing annealing temperature within a temperature profile helped to generate new polymorphisms within flax microsatellite patterns especially with primer 3PCT2. Using this primer we succeeded in generating new polymorphic bands after increasing annealing temperature from 55 °C to 60 °C, and to 65 °C. A cluster analysis of flax cultivars was performed based on microsatellite data. The core group of eight flax cultivars was clustered into two homogeneous subclusters. A lower level of cultivar clustering within subclusters was not detected.