MicroRNAs (miRNAs) are important post-transcriptional regulators for gene expression. Currently, all of the characterized miRNAs are assumed to be transcribed exclusively from the nuclear genome. We characterize a novel microRNA which is solely transcribed from the mitochondrial genome, what we refer to as a mitochondrial microRNA (mito-miRNA). We investigate the effects that single nucleotide polymorphisms (SNPs), in the pre-miRNA region of the candidate mitochondrial miRNA, have on levels of mature miRNA across various ethnicities and families. Our findings potentially present new mechanisms for mitochondrial diseases as well as possible negative effects in gene regulation caused from SNPs in mitochondrial DNA (mtDNA). This supports the increasing need to identify these mtDNA variants in an accurate manner, especially since heteroplasmy of mtDNA can exist in single-cell levels. The essential role of mtDNA for mammalian life is clear due to its role in bioenergetics and protein synthesis. Disruption of these vital biological functions are often caused by mutations and have been linked to many diseases ranging from neurodegeneration to cancer. To better understand the relationship between these mutations and diseases, as well as mito-miRNAs, we develop a more high-resolution and error-free technique named SCAMP-seq (Single-Cell Analysis of Mitochondrial DNA Polymorphisms Sequencing). This paper details SCAMP-seq – an innovative strategy to purify, tag, amplify, and sequence each copy of full-length mtDNA in single cells. We suggest a novel strategy to both remove contaminants and uniquely barcode each mtDNA molecule within a single cell to analyze mtDNA mutations with high-throughput and accuracy.