Mitochondrial DNA (mtDNA) large-scaled deletions have been reported in a broad spectrum of human health conditions. Petite-postive yeast species, Saccharomyces cerevisiae, offers a powerful model to study mtDNA loss, which can be monitored by quantifying the frequency of spontaneously-formed respiratory-deficient (i.e “petite”) colonies in a population. The natural genetic variants contributing to mtDNA loss in yeast presently remain in unmapped territory. By investigating the petite frequency of a subset of haploid derivatives of S. cerevisiae natural isolates, we learned that petite frequency is a variable and heritable trait. We also employed a novel collection of yeast strains harboring unique mito-nuclear genome combinations to determine that petite frequency is influenced by both nuclear and mitochondrial genetic variants, and by mt-n epistatic interactions to a lesser extent. In pursuit of unravelling the mechanism of mtDNA loss in yeast, we also looked into the rate of homologous recombination, which has been proposed to be the predominant mechanism for generating mtDNA deletions in yeast. Our preliminary data suggested that petite frequency positively correlated with growth rate, which leads us to hypothesize that fast growing yeast strains require higher mtDNA replication, hence higher rate of homologous recombination in mtDNA. We sought to investigate if high petite frequency can be explained by high rate of mtDNA recombination. Using yeast strains with diverged nuclear genomes paired with a mitochondrial background containing an ARG8m auxotrophic reporter gene, we found that the rate of homologous recombination, specifically the rate of mtDNA deletion mediated via direct repeats at the COX2 locus, is not correlated with the petite frequency in these yeast strains. Simultaneously, we are developing strategies to map the genetic basis underpinning petite frequency, in hopes of better understanding the mechanism behind spontaneous mtDNA loss in S. cerevisiae.