BACKGROUND: The liver fluke Fasciola hepatica is one of the most important endoparasites in domestic ruminants worldwide and can cause considerable economic losses. This study presents the first population genetic analysis of F. hepatica in Germany and aims at providing new insights into genetic diversity and population structure. METHODS: A total of 774 liver flukes, collected from 60 cows of 17 herds and 13 cows of unknown herd origin, were subjected to comparative analysis of two mitochondrial genes (cox1 and nad1), one nuclear region (internal transcribed spacer (ITS)-1) and eight nuclear microsatellite markers. In addition, individual fluke measurements allowed comparison of morphometric differences between genotypes. RESULTS: The nuclear ITS-1 region showed minimal variability, with 772 of 774 flukes having identical sequences, while the mitochondrial sequences revealed a high genetic diversity, with 119 distinct haplotypes, a mean haplotype diversity (Hd) of 0.81 and a mean nucleotide diversity (π) of 0.0041. Mitochondrial phylogenetic analysis identified two clusters with no clear association with the host or farm of origin. In the microsatellite analysis, all eight loci were highly polymorphic, with a mean allele frequency of 19.0 and a mean genotype frequency of 73.5 per locus. A total of 500 unique multilocus genotypes (MLGs) were found across all fluke samples, indicating that 68.5% of all genotypes were unique. A mean expected heterozygosity of 0.71 suggested a high potential for adaptability and the number of migrants (Nm = 3.5) indicated high gene flow between farms. Population structure analysis based on microsatellite data revealed that flukes from two farms differed genetically from the others. Linear mixed model results revealed that fluke length differed significantly between the two mitochondrial clusters, although it should be noted that fluke age could not be considered in the analyses. CONCLUSIONS: Fasciola hepatica in German dairy farms showed high genetic diversity and gene flow. The differences in population structure identified by mitochondrial sequences compared with microsatellite loci highlight the benefits of analysing genetic markers of different origins. This is the first study to correlate fluke morphometry measurements with genetic markers, indicating that the identified markers can influence fluke size.