The yeast Kluyveromyces marxianus is a thermotolerant yeast with interesting physiological properties that make it interesting producer microorganism for different biotechnical processes. In this work, gene targeting fidelity in the constructed strain of yeast K. marxianus was determined. In the first step, this yeast has been transformed with the linear NatBAV DNA fragment containing a selectable coNat marker responsible for resistance to the antibiotic nourseothricin, whose random integration in the genome was confirmed by Southern blotting. In the second step, strain that contained integrated NatBAV fragment in the genome has been transformed with the linear HygBAV fragment containing coHyg gene responsible for antibiotic resistance to hygromycin, with flanking regions homologous to the NatBAV fragment on either side of the selective marker. As a result of successful gene targeting, the genomic region is replaced with the sequence on the transforming DNA by homologous recombination. After selection on a medium with the antibiotic hygromycin, the obtained transformants have been replicated to the medium with nourseothricin and in 70 % of the transformants the coNat gene was replaced with the coHyg gene. This fidelity was achieved with relatively long lateral homologies (1,4 and 2,5 kb) and based on the results it was found that gene targeting in K. marxianus was significantly more successful than in most other nonconventional yeast species.