Aim Propagule size and ecological requirements are believed to be major factors influencing the passive dispersal of free-living terrestrial protists. We compared the colonization potential of three closely related testate amoeba species (Assulina muscorum, A. seminulum, A. scandinavica, ranging from 40 to 100 μm in length). Location Europe. Methods We collected individual Assulina species cells from Sphagnum peatlands across Europe. We sequenced a 550-bp fragment of the mitochondrial cytochrome c oxidase subunit I gene (COI) to estimate the within-species variability, as a proxy for gene flow. We reviewed existing ecological and palaeoecological data to assess the ecological tolerance of Assulina species and how rapidly they colonized developing peatlands. Results We obtained COI sequences for 30 individuals of A. seminulum from eleven sites, for 39 of A. muscorum from six sites, and for six of A. scandinavica from two sites. We observed three haplotypes for A. seminulum and two for A. muscorum, often co-existing in the same sites. The sequences of A. scandinavica from the two sites were identical. Assulina muscorum and A. seminulum haplotypes varied by only 1–2 nucleotides, resulting in >99.5% similarity. Genetic diversity within A. seminulum was higher than that within A. muscorum. Ecological and palaeoecological records showed that A. muscorum was much more frequent and abundant than A. seminulum, and had a somewhat broader ecological tolerance for pH, moisture and water-table depth. Assulina muscorum always appeared early during the developmental history of peatlands, either before or simultaneously with A. seminulum. Main conclusions The lack of genetic structure observed with a variable marker such as COI suggests high gene flow between the sites and thus rapid transport (at an evolutionary scale) over large distances, in agreement with the palaeoecological records. Thus, geographical distance alone does not seem to prevent the dispersal of testate amoebae, at least within Europe. Nevertheless, genetic diversity was significantly lower within A. muscorum than within A. seminulum, suggesting that its smaller size and abundance and/or broader ecological tolerance influence its effective dispersal capacity. These results are in agreement with the often earlier colonization of peatlands by A. muscorum and its broader ecological tolerance.