Background/Objectives: The black grouse (Lyrurus tetrix) is experiencing a rapid decline across much of Europe. In Poland, the Carpathians and Sudetes constitute two of the last important strongholds of this endangered species; however, the genetic relationships between populations inhabiting these mountain systems remain poorly understood. Methods: A total of 579 non-invasive samples were collected in the Jizera Mountains and Giant Mountains (Sudetes), as well as in the Tatra Mountains and the Orava–Nowy Targ Basin (Carpathians). Microsatellite genotyping at nine loci identified 122 individuals. Genetic diversity and population genetic structure were assessed using F-statistics, AMOVA, STRUCTURE, PCoA and DAPC. Results: Significant genetic differentiation was detected among populations, with substantially lower differentiation within the mountain regions than between the Sudetes and Carpathians. Analyses consistently identified two major genetic groups corresponding to the Sudetes and Carpathians. The highest genetic diversity was recorded in the Tatra Mountains. Some individuals from this population displayed genetic signatures consistent with possible connectivity to neighbouring populations outside the study area, although this hypothesis requires testing with additional reference samples from these populations. Conclusions: The results demonstrate strong genetic structuring of black grouse populations in southern Poland and suggest limited contemporary gene flow between the Sudetes and Carpathians mountain ranges. The observed differentiation supports treating the Sudeten and Carpathian populations as separate Management Units. The Tatra Mountains constitute an important reservoir of genetic diversity and may play a key role in maintaining genetic connectivity within the Carpathian population.