Mitochondrial heterogeneity is the presence of two or more types of mitochondrial (mt)DNA in the same individual/tissue/cell. It is closely related to animal health and disease. ND2 is a protein-coding gene in mtDNA, which participates in mitochondrial respiratory chain and oxidative phosphorylation. In previous studies, we observed that the mt.A5703T and mt.T5727G sites in the ND2 gene were the heteroplasmic variation sites. We used pyrophosphate sequencing technology to examine chicken mt.A5703T and mt.T5727G heteroplasmic sites in the ND2 gene, in different tissues and at different development stages in chickens. We also investigated whether nutritional factors could affect the mt.A5703T and mt.T5727G heteroplasmy. Our results showed that chicken mt.A5703T and mt.T5727G heteroplasmy had clear spatio-temporal specificities, which varied between tissues/development stages. The mtDNA heterogeneity was relatively stable upon nutrition intervention, 30% dietary energy restriction (from 18 to 48 days old) and different types of dietary fats (at 5% concentration, from 1 to 42 days old) did not change the breast muscle heteroplasmy of broilers at the mt.A5703T and mt.T5727G sites. In addition, multiple potential heteroplasmic sites were detected by clone sequencing in the ND2 region, which potentially reflected abundant heteroplasmy in the chicken mitochondrial genome. These results provide an important reference for further research on heteroplasmy in chicken mitochondria.