Background: The genetic diversity information of local cattle and crossbred in South Sulawesi, Indonesia is essential for the establishment of conservation and breeding strategies. This research targeted to evaluate the phylogenetic relationship and genetic diversity of local and crossbred (CB) cattle. Methods: The present research organized at the Animal Laboratory, Professor Nidom Foundation, Surabaya, Indonesia from February to October 2021. The genome was isolated from 20 whole blood samples from seven purebreed belongs to Singosari National Artificial Insemination Center Malang, Indonesia, four crossbred in Gowa and nine crossbred in Sidrap, South Sulawesi, Indonesia. Partial sequences of mtDNA cytochrome oxidase subunit I (COI) gene were amplified employing the polymerase chain reaction technique. Result: This study revealed that a lot of single nucleotide polymorphism (SNP) found in local cattle (Bali and Peranakan Ongole) and CB cattle in Gowa and Sidrap indicated various genetic diversity can be used as a marker candidate and potential to be developed into future research knowing the correlation of the SNP with the growth rate of CB. Thus, this information will be helpful for further strategy in breeding development program.