Aim: Multiple cytomegalovirus (CMV) strain infections correlate with more severe clinical outcomes. Clinical studies could not clarify whether the resistance to CMV defines the number of CMV strains in a patient, or whether viral complemenation may aggravate the prognosis. This study will define the cause-effect relationship between infections by multiple viral strains and cytomegalovirus in vivo growth. Materials and Methods: Singenic mice were infected with attenuated mutants of the mouse cytomegalovirus (MCMV). We compared the growth of attenuated MCMVs in the presence or absence of virulent virus by real-time PCR and infectious virus titration. The role of trans-complementation in MCMV strain interactions in vivo was assessed using MCMV mutants expressing Cre-recombinase or a Cre-inducible GFP gene. Fluorescent microscopy for GFP expression was used to quantify transcomplemenation and results were confirmed by PCR. The relevance of complementation for virus replication was confirmed by in situ hybridization assays specific for different MCMV variants. Results: Coinfection resulted in better replication of attenuated mutants than single infection. The coinfection with the MCMV expressing Cre-recombinase resulted in regular recombination and expression of the GFP gene by the coinfecting virus. Genomes of multiple MCMV variants were found in the same hepatocytes of coinfected animals, and the majority of the cells infected with the attenuated mutant were at the same time infected with the replication competent MCMV. Conclusion: Multiple CMV variants are found in the same cells during in vivo coinfection, facilitating functional complementation and replication of viral variants. Thus, the results indicate that coinfection with multiple CMV strains leads to viral complementation, which may worsen the clinical picture and the prognosis.