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Many rotifers are considered to be cosmopolitan species including Epiphanes senta, which has been reported from all continents including Antarctica. To determine the potential for cryptic speciation in this species, we examined morphology, mating behavior and genetic divergence in three populations: (1) a tropical alpine lake on Mauna Kea (HI, USA), (2) temporary rock pools of the Chihuahuan Desert (TX, USA), and (3) floodplain habitats of the Oder River (Germany). Morphological differences among these populations are restricted to the surface structure of diapausing eggs and the shape of the rami. In crossmating experiments, German males never fertilized females from the Texan population. Texan males mated with hatching females from the German population. However, juvenile mortality among these fertilized females reached 40% and was significantly higher than juvenile mortality after intrapopulational mating (2%). Surviving females did not produce viable diapausing eggs. Hawaiian males readily mated with newborn Texan females, which produced viable offspring. They also copulated with females from the German population, but these females never produced diapausing eggs. Genetic distance among the three populations was considerable, ranging from 10.9 to 12.5% for 648 bp of the mitochondrial cox1 gene. Morphological, reproductive, and genetic differentiation among the studied populations demonstrates E. senta is actually a species complex. Three new species within the E. senta species complex are described.