Mitochondria are important and unique organelles in the cell that are mainly responsible for energy metabolism. However, due to its unique property of having its own DNA, it also is involved in many other cellular processes. Due to this major role of mitochondria in the cell, the organelle also plays important roles in many diseases, including mitochondria-inherited diseases, metabolic diseases, and cancer. Recently, more research has been targeted at elucidating the role of mitochondria and mitochondrial DNA in cancer and how mitochondrial dysfunction can be drug targeted. The work presented here aims to further investigate the role of specific mitochondrial DNA mutations and heteroplasmy in mitochondrial function and cancer traits.Chapter one is a review of the current knowledge of mitochondrial genetics and the involvement of mitochondrial dysfunction in various human diseases. It also summarizes the contemporary knowledge about the role of mitochondria and heteroplasmy in cancer and tumor progression, with an emphasis on breast cancer. Furthermore, it goes into using drugs to target mitochondrial DNA and metabolic dysfunction. Chapter two describes the initial investigation done on a non-pathogenic high heteroplasmy mutation in a colon cancer cell line and its correlation with mitochondrial membrane potential. Even though no association was found between heteroplasmy and membrane potential, it did shift the direction of my investigation toward finding pathogenic non-synonymous mutations that can be segregated into distinct heteroplasmy groups. Chapters three and four of my dissertation focuses on the effects of low and high heteroplasmy of two amino acid changing mutations on mitochondrial function and cancer phenotype in both tripe negative breast cancer cell lines and a cybrid cell line. Using various cancer specific assays, I was able to show that high heteroplasmy influences anchorage-independent growth and for the cybrid cell line, invasiveness as well. When examining differences in mitochondrial function, the high heteroplasmy samples showed a higher oxygen consumption rate than the low heteroplasmy groups. Furthermore, when examining nuclear gene expression changes, there were significant differences between the high vs low group in various cancer promoting and inhibiting pathways, respectively.