Benha line chickens (Line B) were originated by crossing the Egyptian native Golden Montazah cocks with the White Leghorn chickens hens. The main aim of this study was to estimate the genetic characterization of Benha Line Chickens compared to Golden Montazah and White Leghorn chickens as parents. In this study, the genomic DNA of B line, Golden Montazah and White Leghorn chickens were extracted to carry out Polymerase Chain Reaction (PCR) using five specific microsatellite markers (MCW0330, MCW0183, MCW0081, MCW0014 and ADL0278). These markers are specific for egg number, egg shell strength, clutch size, breast muscle weight (BMWT), body weight, egg production rate, heat stress and growth. Further, MCW0330 and MCW0081 primers are specific for resistance of Salmonella and Marek's (MD) diseases. Our results confirm that these specific microsatellite markers are specific for the Benha line chickens except the ADL0278 not specific for Benha line chickens. Further, 26 distinct alleles were detected at the five microsatellite markers. Amongst 26 distinct alleles, Benha Line was carried the largest number of specific alleles at five (20.8%), followed by Golden Montazah with two specific alleles (10.5%). In addition to, the polymorphic information content (PIC) and expected heterozygosity (He) for the three chicken populations were calculated. Benha Line chickens were the highest values of PIC and He per locus which were observed at MCW0183, MCW0330 and ADL0278, respectively, and the corresponding lowest were at MCW0014 and MCW0081, respectively. Moreover, chromosomal analysis was carried out and our results confirm that Benha Line chickens have a diploid number of 78 (2n = 78) chromosomes and the majority are microchromosomes.