establishing adequate utilization of genetic variation in developing livestock breeding programs. Cytochrome oxidase subunit I (COI) gene technique was used in the present study to identify and differentiate the main three Egyptian local goat breeds (i.e. Baladi, Zaraibi and Barki) via DNA barcoding to confirm its species identity and provide valuable DNA sequence source in the nucleotide online database for further studies. Blast (Basic Local Alignment Search Tool) results confirmed samples to be Capra hircus (100%) with no variation among the studied breeds. On the other hand, the Fluorescently Amplified Fragment Length Polymorphism (F-AFLP) technique was applied to assess genetic variation among and within the three breeds for litter size character. F-AFLP analysis of triplicates per breed produced 164 polymorphic loci. At the same time fixed and private bands varied among the three breeds; 47, 17 and 14 bands and 9, 19 and 27 bands for Baladi, Zaraibi and Barki, respectively. Analysis of Molecular Variance (AMOVA) showed 3.8% and 96.1% variance among and within breeds, respectively. Population re-allocation showed that all samples of Baladi breed are outliers, Zaraibi breed one outlier and two hybrids and in Barki breed one hybrid, one outlier and one allocates itself. Private bands in excel filter (using virtual inspection in excel) showed fixed bands of 213bp molecular weight at locus 35 in both Baladi and Zaraibi breeds. These bands considered as genetic marker for litter size trait (i.e. high prolific animals).