Microsatellite markers were employed to determine the genetic diversity and structure of eight goat breeds kept in the Czech Republic (White Shorthaired goat, Brown Shorthaired goat, Alpine, Anglo-Nubian) and Poland (Polish White Improved, Polish Fawn Improved goat, Sandomierska, Toggenburg). Across 398 individuals analyzed using 15 microsatellite markers, a total of 191 alleles were detected, with a mean of 7.108 alleles per locus. The average of observed and expected heterozygosity, fixation index, and Shannon’s information index were 0.684 ± 0.014, 0.696 ± 0.013, 0.014 ± 0.012, and 1.476 ± 0.037, respectively. The mean of overall genetic differentiation (FST) was 0.110, heterozygote deficiency among populations (FIT) was 0.125, and the inbreeding coefficient within the goat population (FIS) was 0.018. Analysis of molecular variance showed that 10% of the total genetic variation was attributed to differences among populations, 3% among individuals within populations, 86% within individuals, and 1% was observed among regions. A phylogenetic dendrogram and principal coordinate analysis (PCoA) revealed three main clusters, while STRUCTURE analysis identified two main gene pools. These findings provide critical insights for future genetic improvement, breeding program optimization, and conservation strategies for local landrace goat breeds, whose genetic resources are pivotal for maintaining livestock biodiversity.