<p>Heteroplasmy is characterized by different mitochondrial haplotypes within the same organism. After evidence of heteroplasmy was found in mitochondrial sequences in <i>Tetragonisca angustula</i>—a bee species of great economic and ecological importance—the aim of this study was to confirm the presence of heteroplasmy in the species, analyse the frequency of synonymous and non-synonymous mutations, and classify them according to their impact on the protein’s tertiary structure. For this purpose, mitochondrial DNA was isolated from nuclear DNA in two <i>T. angustula</i> individuals, a portion of <i>COI</i> was amplified by PCR from pure mitochondrial DNA, and the different mitochondrial haplotypes were recovered through vector cloning in competent bacteria. Our results confirmed the presence of 11 and 17 different mitochondrial haplotypes in the first and second samples analysed, respectively. These different mitochondrial haplotypes are characterized by the predominance of synonymous mutations or mild and moderate non-synonymous mutations, meaning they have little impact on the protein’s tertiary structure. These results strongly support the presence of heteroplasmy in <i>T. angustula</i> and provide some insights into its characterization and inheritance.</p>