100 strains were isolated from traditional Kazak cheese in different parts of Xinjiang Uygur Autonomous Region. Four yeast strains Y-2, Z-5, S-6 and N-5 of producing lipase were screened by RB and Rhodamine B medium and they were identified by 26S rRNA gene D1/D2 and internal transcribed spacers. The results showed that strains Y-2 and Z-5 were belonged to Kluyveromyces marxianus, while S-6 and N-5 belonged to Pichia kudriavzevii and Kluyveromyces lactis, respectively. The lipase producing activities of the 4 strains were determined by p-nitrophenol method, and the K. marxianus Z-5 was the highest, up to 23.00 U/mL. The optimum lipase production conditions of K. marxianus Z-5 were determined as follows: fermentation temperature 30 ℃, pH 7.5, inoculum 2.5 mL/100 mL. The obtained enzymatic activity was 25.99 U/mL.