Abstract. Phromnoi S, Lertwatcharasarakul P, Phattanakunanan S. 2021. Genetic diversity and phylogenetic analysis of Khiew-Phalee chickens based on mitochondrial DNA cytochrome b gene sequences. Biodiversitas 23: 750-756. Khiew-Phalee Chickens (Gallus gallus) is a Thai indigenous breed, locally recognized for its economic value as an important source of revenue generation. As the Khiew-Phalee chicken cannot be accurately identified by morphological characterization, molecular methods for species identification are generally thought to be complementary and more effective than relying on the method alone. Molecular characterization of Khiew-Phalee Chickens was investigated using mitochondrial DNA (mtDNA) cytochrome b (Cyt b) gene analysis. Partial sequences of the 1,037 bp Cyt b gene were determined on 55 specimens of Khiew-Phalee chickens. The breed's genetic diversity was assessed based on the number of polymorphic sites, number of haplotypes, haplotype diversity, nucleotide diversity, and average number of differences. In addition, a neighbor-joining (NJ) haplotype tree was constructed based on Kimura's two-parameter model. Haplotype and nucleotide diversity of Khiew-Phalee chickens were 0.597 and 0.00068, respectively. Five haplotypes were identified from 4 polymorphic sites with polymorphism at nucleotides 501, 507, 543, and 1,095. Phylogenetic analysis with the inclusion of the Cyt b gene nucleotide sequences from Khiew-Phalee chickens and native chickens from the GenBank database showed that Khiew-Phalee chickens belonged to Gallus gallus. It was closest to the indigenous chicken in South East Asia. The phylogenetic tree indicated the genetic variability of the investigated Khiew-Phalee chicken populations. This study may help future researchers and livestock breeders to design a breeding program based on a better understanding of the genetic diversity and history of indigenous breeds.