Copyright © 2019 the American Physiological Society. Hyperoxia-induced oxidant stress contributes to the pathogenesis of bronchopulmonary dysplasia (BPD) in preterm infants. Mitochondrial functional differences due to mitochondrial DNA (mtDNA) variations are important modifiers of oxidant stress responses. The objective of this study was to determine whether mtDNA variation independently modifies lung development and mechanical dysfunction in newborn mice exposed to hyperoxia. Newborn C57BL6 wild type (C57n/C57mt, C57WT) and C3H/HeN wild type (C3Hn/C3Hmt, C3HWT) mice and novel Mitochondrialnuclear eXchange (MNX) strains with nuclear DNA (nDNA) from their parent strain and mtDNA from the other-C57MNX (C57n/ C3Hmt) and C3HMNX (C3Hn/C57mt)-were exposed to 21% or 85% O2 from birth to postnatal day 14 (P14). Lung mechanics and histopathology were examined on P15. Neonatal mouse lung fibroblast (NMLF) bioenergetics and mitochondrial superoxide (O2-) generation were measured. Pulmonary resistance and mitochondrial O2-generation were increased while alveolarization, compliance, and NMLF basal and maximal oxygen consumption rate were decreased in hyperoxia-exposed C57WT mice (C57n/C57mt) versus C57MNX mice (C57n/C3Hmt) and in hyperoxia-exposed C3HMNX mice (C3Hn/C57mt) versus C3HWT (C3Hn/C3Hmt) mice. Our study suggests that neonatal C57 mtDNA-carrying strains have increased hyperoxia- induced hypoalveolarization, pulmonary mechanical dysfunction, and mitochondrial bioenergetic and redox dysfunction versus C3H mtDNA strains. Therefore, mtDNA haplogroup variation-induced differences in mitochondrial function could modify neonatal alveolar development and BPD susceptibility.