Mitochondrial DNA copy number (mtDNA-CN) is a biomarker for mitochondrial dysfunction associated with several diseases. Previous genome-wide association studies (GWAS) have been performed to unravel underlying mechanisms of mtDNA-CN regulation. However, the identified gene regions explain only a small fraction of mtDNA-CN variability. Most of this data has been estimated from microarrays based on various pipelines. In the present study we aimed to (1) identify genetic loci for qPCR-measured mtDNA-CN from three studies (16,130 participants) using GWAS, (2) identify potential systematic differences between our qPCR derived mtDNA-CN measurements compared to the published microarray intensity-based estimates, and (3) disentangle the nuclear from mitochondrial regulation of the mtDNA-CN phenotype. We identified two genome-wide significant autosomal loci associated with qPCR-measured mtDNA-CN: at HBS1L (rs4895440, p = 3.39 × 10 ) and GSDMA (rs56030650, p = 4.85 × 10 ) genes. Moreover, 113/115 of the previously published SNPs identified by microarray-based analyses were significantly equivalent with our findings. In our study, the mitochondrial genome itself contributed only marginally to mtDNA-CN regulation as we only detected a single rare mitochondrial variant associated with mtDNA-CN. Furthermore, we incorporated mitochondrial haplogroups into our analyses to explore their potential impact on mtDNA-CN. However, our findings indicate that they do not exert any significant influence on our results.