Background: The present study was carried out for genetic characterization and assessment of evolutionary status of native cattle breeds of Tamil Nadu, India. Methods: Complete mitochondrial genome sequence of 15 pooled samples belonging to five cattle breeds of Tamil Nadu was carried out for the first time using Illumina platform. These mitogenomes were utilized in the present investigation to study the mitochondrial diversity. using suitable statistical tools. Result: The sequences encompassed 16,338 to 16,340 nucleotides with 273 variants. Low degree of genetic divergence and polymorphism was observed in the population. Higher rate of migrants (11.77) between populations caused increase in gene flow. FST value of -0.04435 revealed low level of genetic differentiation between cattle breeds of Tamil Nadu. A significant negative Tajima’s D (P less than 0.05) for all the Bos indicus cattle indicates operation of purifying selection and population expansion among the genetic groups under study. Maximum likelihood reconstruction using complete mitochondrial sequences of the present study combined with previously reported sequences of Nellore cattle and eight Bos taurus cattle breeds illustrated close genetic relationship among the Bos indicus cattle under study with a clear demarcation from the Bos taurus cattle indicating higher genetic divergence between the two lineages. This finding was also supported by multi-dimensional scaling. Analysis of molecular variance revealed no differentiation between cattle breeds of Tamil Nadu.