In the current work, the genetic variety of Gallus gallus from four sites was evaluated using large mitochondrial rRNA (16S rRNA). Samples were gathered from four different places within the Egyptian Governorate of Qena; Naga Hammadi City, Qena City, El-Taramsa Village and Qus City. The nucleotide lengths obtained from sequencing 16S rRNA gene of the mitochondria in the four sites varied from 547 base pairs (bp) to 565 bp. The 16S rRNA partial nucleotide sequences were entered into GenBank with accession numbers (PP422403 - PP422410). The findings show that the 547 bp nucleotide sequences found in Naga Hammadi City are the shortest. 569 bp made up the final alignments; of them, 566, three, and two were conserved sites, variable sites, and parsimony informative sites, respectively. Compared to the C+G content, the average A+T content was higher at 52.98%. Low genetic similarity was observed between Naga Hammadi and Qena Cities, this may be due to many factors like geographical and ecological conditions