RNA viruses mutate rapidly due to their error-prone replication and are one of the fastest-evolving biological entities. Mixed infections are common in nature and interaction between viruses in mixed infection can represent a change in selective pressures, in comparison to a single infection, and might have an effect on the evolution of viruses present in mixed infection. Pepino mosaic virus (PepMV) and Potato virus Y (PVY) are well-known plant pathogens, causing loss of tomato and potato yields, respectively. We were interested whether mixed infections of PepMV and PVY affect their evolution or whether due to direct or indirect interactions between viruses in a mixed infection, the dynamics of the evolution of viral populations in plants can change. In order to test this, we used an experimental evolution approach in Nicotiana benthamiana plants. Several evolutionary lines of mixed and single infections of PepMV and PVY were established and serial passages of virus populations onto non-infected plants were performed. Using quantitative polymerase chain reaction we monitored viral titers in evolutionary lines of mixed and single infections during the experimental evolution and observed symptoms of inoculated plants in different passages. Mixed and single infections of PepMV and PVY was successfully established in Nicotiana benthamiana plants. Disease symptoms were more pronounced in mixed infections, when compared with single infections. Multiplication and systemic infection formation of PVY and PepMV in N. benthamiana occurred at different pace for each of the viruses. We observed slight changes in viral titers during the course of experimental evolution and we hypothesise those could be attributed to the effect of genetic drift and/or adaptation of viruses in mixed infection.