Leber's hereditary optic neuropathy (LHON), the most frequent mitochondrial disorder, is mostly due to three mitochondrial DNA (mtDNA) mutations in respiratory chain complex I subunit genes: 3460/ND1, 11778/ ND4 and 14484/ND6. Despite considerable clinical evidences, a genetic modifying role of the mtDNA haplogroup background in the clinical expression of LHON remains experimentally unproven. We investigated the effect of mtDNA haplogroups on the assembly of oxidative phosphorylation (OXPHOS) complexes in transmitochondrial hybrids (cybrids) harboring the three common LHON mutations. The steady-state levels of respiratory chain complexes appeared normal in mutant cybrids. However, an accumulation of low molecular weight subcomplexes suggested a complex I assembly/stability defect, which was further demonstrated by reversibly inhibiting mitochondrial protein translation with doxycycline. Our results showed differentially delayed assembly rates of respiratory chain complexes I, III and IV amongst mutants belonging to different mtDNA haplogroups, revealing that specific mtDNA polymorphisms may modify the pathogenic potential of LHON mutations by affecting the overall assembly kinetics of OXPHOS complexes.