The aim of the study was to develop the system of polymerase chain reaction (PCR)-based markers for the assessment of genetic diversity and population genetic studies of Gentiana lutea L. as well as to determine the utility of two indices of marker informativeness. The informativeness was determined for 40 PCR primers of different types (random amplified polymorphic DNA, inter simple sequence repeat, inter-retrotransposon amplified polymorphism, resistance gene analog polymorphism and conserved DNA-derived polymorphism markers) by evaluating discriminating power (DL) and resolving power (Rp) in a sample of 30 plants from two populations. Analysis of correlation between the index value and the number of differentiated pairs of genotypes in the given sample revealed that DL is more efficient than Rp; therefore, we selected primers based on the DL value. In total, 12 primers with the largest values of DL were chosen. Analysis of genetic relationship among 86 plants from six populations showed that the number of bands produced by the three of selected primers was sufficient to give average bootstrap support across six key nodes in the dendrogram higher than 85%, while using six of the primers resulted in average bootstrap value exceeding 99%. Thus, a minimal set of three to six selected primers are sufficient for a quick assessment of genetic diversity of G. lutea populations, depending on the sample size and degree of differentiation between populations, while the rest of the primers with DL values above 0.8 may be used for ecogenetic surveys. Preliminary results obtained with selected primers indicate the moderate level of genetic variation within the species and significant differentiation among individual populations.