Tobacco mosaic virus (TMV) derivatives are explored currently extensively with regard to nanotechnological applications. Since certain technically desired TMV mutants may not be accessible from plants, the utility of the fission yeast Schizosaccharomyces pombe for heterologous production of TMV coat protein (CP) variants was explored, including wild-type (wt) CP and two genetically engineered mutants: TMV-CP-His(6) containing a C-terminal hexahistidine (His(6)) tag, and TMV-CP-E50Q with enhanced lateral CP subunit interactions. After establishing expression clones and protocols for enrichment of the CP variants, their ability to reconstitute TMV-like nanostructures in the presence or absence of RNA was tested in comparison with the corresponding plant-derived CP variants, which were expressed from infectious TMV constructs. Both TMV-CP-E50Q and TMV-CP-wt yielded TMV-like rods, irrespective of the proteins' source. In contrast, His-tagged CP from plants produced only short rods in an inefficient manner, and no rods at all when expressed in yeast. This study introduces a novel approach to produce assembly competent TMV CP, but also demonstrates its limitations.