Vaccines against porcine circovirus 2 (PCV2) are now widely used to control the diseases caused by the virus. Although the vaccines protect pigs against the disease, they do not lead to sterilizing immunity and therefore infections with PCV2 continue in farms. It is expected that, due to its high evolutionary rate, PCV2 can adapt quickly to environmental pressures such as vaccination. The goal of this study was to elucidate the molecular variation of PCV2 in relation to vaccination. PCV2 variability was investigated from samples of infected pigs from five farms where vaccination had never been applied and two farms where pigs had been vaccinated for at least 2 years. For the genetic analysis, full PCV2 genomes were amplified and subsequently pooled by vaccination status from serum of eight vaccinated, infected pigs and 16 non-vaccinated, infected pigs. Variability of viral populations was quantified using next-generation sequencing and subsequent bioinformatics analysis. The number of segregating sites was similar in the non-vaccinated (n = 109) and vaccinated pools (n = 96), but the distribution of these sites in the genome differed. Most notably, in the capsid gene, the number of segregating sites was observed only in the non-vaccinated population. Based on the structural analysis, it is expected that some low-frequency amino acids result in biologically low-fit viruses. On the contrary, D294 in replicase represents a novel amino acid which was dominant and unique in the vaccinated pool. This work showed that variable PCV2 populations were circulating in commercial farms, and that this variability was different in samples obtained from vaccinating and non-vaccinating farms. © 2014 The Authors.