Mumps virus is the causative agent of mumps disease, of which humans are the only natural host. Mumps infection is vaccine-preventable. Mumps vaccines are live viruses attenuated by serial passage on cell substrates or in embryonated eggs. They are known to vary in their effectiveness, degree of attenuation and adverse event profile. The Jeryl Lynn mumps virus is the most commonly used vaccine strain. Two related commercial mumps vaccines, which originate from the same attenuated strain (Jeryl Lynn-5) but have different efficacies, were investigated. Jeryl Lynn-Canine Kidney (JL-CK), produced on primary canine kidney cells is less effective than RIT 4385 which is produced on chicken embryo fibroblasts. JL-CK and RIT 4385 could be distinguished by a number of in vitro and in vivo properties. JL-CK produced heterogeneous, generally smaller plaques than RIT 4385, but gave 100 fold higher viral titres when grown on Vero or MDCK cells and produced a higher degree of hydrocephalus in the neonatal rat brain. Sequencing of the JL-CK virus identified 14 regions of heterogeneity throughout the genome. Plaque isolation demonstrated the presence of five mumps virus variants encompassing these mutations in the JL-CK vaccine. One mutation was associated with a small plaque phenotype, the effects of the others either in vitro or in vivo were less clear. Only 4 % of the JL-CK vaccine population corresponded with the parent Jeryl Lynn-5 strain. Deep sequencing of JL-CK and virus replicating in cell lines and neonatal rat brains showed that propagation in vitro or in vivo altered the population dramatically. The data presented in this study suggest that growth of JL-CK in primary canine kidney cells resulted in the selection of a mixture of mumps virus variants that have different biological properties compared to the parent Jeryl Lynn-5 virus.