Kluyveromyces marxianus is a thermotolerant, ethanol-producing yeast that requires oxygen for efficient ethanol fermentation. Under anaerobic conditions, glucose consumption and ethanol production are retarded, suggesting that oxygen affects the metabolic state of K. marxianus. Mitochondria require oxygen to function, and their forms and number vary according to environmental conditions. In this study, the effect of anoxia on mitochondrial behavior in K. marxianus was examined. Under aerobic growth conditions, mitochondria-targeted GFP exhibited a tubular and dotted localization, representing a typical mitochondrial morphology, but under anaerobic conditions, GFP localized in vacuoles, suggesting that mitophagy occurs under anaerobic conditions. To confirm mitophagy induction, the ATG32, ATG8, ATG11 and ATG19 genes were disrupted. Vacuolar localization of mitochondria-targeted GFP under anaerobic conditions was interrupted in the Δatg32 and Δatg8 strains but not the Δatg11 and Δatg19 strains. Electron microscopy revealed mitochondria-like membrane components in the vacuoles of wild-type cells grown under anaerobic conditions. Quantitative analyses using mitochondria-targeted Pho8 demonstrated that mitophagy was induced in K. marxianus by anoxia but not nitrogen starvation. To the best of our knowledge, this is the first demonstration of anoxia-induced mitophagy in yeasts. © The Author(s) 2020. Published by Oxford University Press on behalf of FEMS.