Chlamydomonas reinhardtii and Saccharomyces cerevisiae are currently the two microorganisms in which genetic transformation of mitochondria is routinely made. The generation of a large variety of defined alterations as well as the insertion of ectopic genes in the mitochondrial genome (mtDNA) are possible, especially in yeast. Biolistic transformation of mitochondria is achieved through the bombardment of microprojectiles coated with DNA, which can be incorporated into mtDNA thanks to the highly efficient homologous recombination machinery present in S. cerevisiae and C. reinhardtii organelles. Despite a low frequency of transformation, the isolation of transformants in yeast is relatively quick and easy since several natural or artificial selectable markers are available, while the selection in C. reinhardtii remains long and awaits for new markers. Here we describe the materials and technics used to perform biolistic transformation, in order to mutagenize endogenous mitochondrial genes or insert novel markers into mtDNA. Although alternative strategies to edit mtDNA are being set up, so far insertion of ectopic genes relies on the biolistic transformation technics.