Over the past years, remarkable progress has been made in understanding structure and function of genes and chromosomes at the molecular level. Recently, this progress has been made in understanding the organization of the human genome at level of its DNA sequences. Highly polymorphic variable number of tandem repeats [VNTR] provide excellent means for discrimination between individuals. It is a Mendelian monogenic trait that exists in the population in at least two phenotypes neither of which occurs with a frequency of less than 1%-2%. D1S80 locus one of VNTR is located on chromosome 1p36. 1-36.3 and has a core sequence of 16 base pair [bp] that can repeats at range from 14 to over 41 times. It has no known function to regulate an expression of a gene and it shows high level of genetic variation useful for forensic and paternity work. The aim of this study was to investigate the allele and genotype frequency distribution of D1S80 locus in Egyptian population. The usefulness of this genetic marker for paternity testing was also evaluated. The polymorphism of the D1S80 locus has been analyzed in a population sample of 150 unrelated individuals in Upper Egypt, northern Egypt, and Bedwin. The study demonstrates that the locus is highly polymorphic in Egyptian population; there were 26 different D1S80 alleles and 50 distinct genotypes in the studied sample. Allele 18 [f = 0.223] and allele 24 [f=0.197] were the most common alleles. The distribution of the observed genotypes showed departure from Hard-Weinberg expectation. The heterozygosity, probability of exclusion, and discrimination probability estimates demonstrate the usefulness of this locus for paternity purposes.