Nonconventional yeasts include all yeast species except Saccharomyces cerevisiae and Schizosaccharomyces pombe, the two most commonly used species in industry. Many nonconventional yeasts have the ability to metabolize a wide range of substrates, produce specific relevant secondary metabolites, and often show greater resistance to stressful conditions such as high temperatures, osmotic pressure, the presence of toxic substances or extreme pH values. One of the most biotechnologically significant characteristics of the nonconventional yeast Spathaspora passalidarum is that, thanks to specific genes and biosynthetic pathways, it can ferment xylose, the sugar present in lignocellulosic biomass. To harness the potential of nonconventional yeasts and improve their characteristics, it is essential to be able to genetically modify them. Therefore, the aim of this work is to transform the yeast Spathaspora passalidarum with different transforming DNA molecules and to examine the activity of replication origins from various yeast species using plasmid vectors. The transformation process was carried out using the electroporation method previously developed for the yeast Brettanomyces bruxellensis, and the obtained results showed that the highest transformation efficiency is achieved with the plasmid containing the replication origin from the yeast Scheffersomyces stipitis.