Plant virus diversity and the spatial distribution of viral strains or isolates are studied at many different scales: global, regional, local, and even within a single host in different organs or tissues. However, one level that has been totally lacking at the extremity of this scale is that of the single cell. The technical difficulties involved in isolating individual cells from infected plants, and the lack of an efficient diagnostic procedure allowing the specific detection of viral sequences with no major contamination from other cells, have precluded such single cell analysis to date. This paper describes the preparation of protoplasts from plants infected with Cauliflower mosaic virus (CaMV), and their decontamination and separation using a technique requiring no specialised equipment. Efficient single-cell nested-PCR procedures (both standard and high-resolution-melting) were developed to allow efficient amplification and analysis of viral sequences from isolated single cells. Moreover, the specific identification of two CaMV variants in different cells demonstrated a very low level of cross-contamination. This technique paves the way for the future development of numerous applications of broad interest in the study of viral diversity and population genetics of plant viruses at the cellular level.