Chicken mitochondrial DNA is a circular molecule comprising ~16.8 kb. In this study, we used next-generation sequencing to investigate mitochondrial heteroplasmy in the whole chicken mitochondrial genome. Based on heteroplasmic detection thresholds at the 0.5% level, 178 cases of heteroplasmy were identified in the chicken mitochondrial genome, where 83% were due to nucleotide transitions. D-loop regionwas hot spot region for mtDNA heteroplasmy in the chicken since 130 cases of heteroplasmy were located in these regions. Heteroplasmy varied among intraindividual tissues with allele-specific, position-specific, and tissue-specific features. Skeletal muscle had the highest abundance of heteroplasmy. Cases of heteroplasmy at mt.G8682A and mt.G16121A were validated by PCR-restriction fragment length polymorphism analysis, which showed that both had low ratios of heteroplasmy occurrence in five natural breeds. Polymorphic sites were easy to distinguish. Based on NGS data for crureus tissues, mitochondrial mutation/heteroplasmy exhibited clear maternal inheritance features at the whole mitochondrial genomic level. Further investigations of the heterogeneity of the mt.A5694T and mt.T5718G transitions between generations using pyrosequencing based on pedigree information indicated that the degree of heteroplasmy and the occurrence ratio of heteroplasmy decreased greatly from the F0 to F1 generations in the mt.A5694T and mt.T5718G site. Thus, the intergenerational transmission of heteroplasmy in chicken mtDNA exhibited a rapid shift toward homoplasmy within a single generation. Our findings indicate that heteroplasmy is a widespread phenomenon in chicken mitochondrial genome, in which most sites exhibit low heteroplasmy and the allele frequency at heteroplasmic sites changes significantly during transmission events. It suggests that heteroplasmy may be under negative selection to some degree in the chicken.