Conservation of the intraspecific genetic diversity of trees is crucial for long-term forest sustainability, especially in the expected scenario of climate change. Larix decidua is an excellent example of a very common species for which better knowledge of the population genetic structure is needed. The aim of this study was to examine the molecular genetic variability of L. decidua in the Alps and Western Carpathians with adjacent areas using simple sequence repeat (SSR) markers. Low to moderate levels of genetic differentiation were observed among the study populations across the native distribution area of L. decidua. Based on AMOVA and PCoA, we revealed a significant correlation between genetic and geographical distances in the whole dataset. The group of analysed populations of Carpathian larch from Eastern Poland, the area of the Jeseníky Mountains in the Czech Republic and the Tatra Region in Slovakia is relatively homogenous with regard to genetics. The FST values between these populations did not exceed 0.05, indicating low genetic differentiation. In addition, the population from the Eastern Alps could be assigned to this group. A certain level of relationship to Carpathian populations was also observed in the populations from the Northern Alps. In contrast, more significant genetic differences were proven between the individual regions within the analysed populations of Alpine larch from the Southern Alps, including the Maritime Alps, Central Alps, and north-eastern and eastern borders of the Alps. The FST values between the individual populations from different Alpine regions indicated, in most cases, a medium genetic divergence (0.025–0.112).Further research is needed to obtain more detailed information about the genetic variability of local native populations of L. decidua and their geographical determination, especially in the area of the Alps.