Mycoplasma bovis is one of the major etiologic agents of bovine mycoplasmosis, causing diseases such as pneumonia, mastitis and arthritis. This bacterium is spread worldwide and leads to enormous economic loss for both the dairy and the beef industries, especially in North America. Despite the large amount of research performed to better understand the mechanisms involved in the pathogenicity of this mycoplasmal species, little knowledge is available. Furthermore, there is no cell system, which allows to link the virulence of strains with their clinical or pathological features. Phenotypic differences among strains were previously shown but none of these studies could associate a specific pathological picture or severity of disease with a particular strain. In Switzerland, M. bovis infections were mainly associated with pneumonia and subclinical mastitis. The emergence of severe M. bovis-mastitis cases was observed in 2007-2008. New and old Swiss isolates were analyzed by Multi-Locus Sequence Typing and two major lineages were identified. Recent strains cluster with ST5, while all strains isolated before 2007 cluster with ST17. In order to experimentally assess field observations, bovine cell infection assays were developed. These in vitro assays were tested to measure cell invasion and intracellular replication of M. bovis using the gentamicin protection assay. Cell invasion and intracellular replication was shown for both phylogenetic lineages in all tested cell types. Additionally, the strain of M. bovis selected as a representative strain of the old lineage revealed a doubled intracellular generation time in primary bovine epithelial mammary gland cells compared to the new lineage. This is the first experimental evidence linking a specific phylogenetic cluster of M. bovis to differential virulence towards mammary gland cells and to the rise of severe mastitis cases.