The natural consequences of introducing alien species can be significant. This is particularly a concern where the taxa have an invasive nature of spreading or in those that freely crossbreed with native species. The hybridization process may lead to impoverishment or even loss of the native gene pool. This is especially dangerous in unique areas that stand out due to their special natural characteristics, such as the Tatra National Park. The determination of the scale of occurrence of alien larch species in the national park and the evaluation of the genetic diversity of the native population is crucial for the conservation of genetic resources and strictly adheres to the latest conservation genetics trends. We evaluated the possibility of effective use of molecular markers for taxonomic identification of the native European larch (Larix decidua Mill.), as well as the alien Japanese larch (Larix kaempferi [Lambert] Carriere) and the hybrid form (Larix × eurolepis Henry). Microsatellite markers were used to analyse the genetic diversity of individuals identified as European larch from natural refuges and artificial plantings. Of the 148 trees analysed, 105 were identified as the European larch, 38 as Japanese larch, and five as hybrids. The analysis of the molecular variability of two European larch groups of indigenous and artificial origin showed comparable level of diversity. This study confirmed the effectiveness of the use of selected molecular markers in identification of larch species, which is difficult based on morphological traits. The results indicate the possibility for the effective use of genetic tools in the creation of protection programmes, especially for naturally valuable sites, based on genetic taxonomic identification and richness verification of protected gene pools.