The aim of this study was to quantify the genetic variation of the Iraqi local cattle breeds in the Middle Al-Furat in Iraq. Ten microsatellite markers were used to estimate parameters of genetic variation. Genotypes of 60 animals from each of the three cities (Babylon, Karbala, and Najaf) were analysed for genetic diversity and genetic distance. The results showed overall mean observed (Na) and expected numbers (No) of alleles were found to be (7±1.56), and (5.22±1.31), respectively. The mean Shannon’s information index (I) and Polymorphism information content (PIC) values were found to be 1.75 ± 0.24 and 0.70 ± 0.02, respectively. The mean observed (Ho) and expected (He) heterozygosity per locus values were found to be 0.56 ± 0.10 and 0.80 ± 0.04, respectively. The mean expected heterozygosity (Nei) and fixation index (Fis) were found to be 0.79 ± 0.04 and 0.28 ± 0.02, respectively. The mean F-statistics (FIS, FIT, and FST) and were found to be 0.272, 0.293 and 0.029, respectively.