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To elucidate the spectra of Prototheca species and P. zopfii genotypes in milk samples from mastitis affected cattle, 200 Prototheca-isolates of 57 German dairy cattle barns were analyzed by species- and genotype- specific PCR and RFLP analysis. The investigations showed that 177 (88.5%) of all isolates represented P. zopfii genotype 2, 21 (10.5%) isolates represented P. blaschkeae and two (1.0%) P. zopfii genotype 1. Therefore mainly P. zopfii genotype 2 but also P. blaschkeae act in the epidemiology of mastitis in cattle, whereas the role of P. blaschkeae in development of bovine protothecal mastitis should be further explored in epidemiological studies. In this study P. blaschkeae was demonstrated for the first time in association with bovine mastitis in Germany.