Twenty-eight original chicken microsatellite markers were isolated and characterized to determine their utility as cross-reactive markers for comparative genetic mapping in the order Galliformes. Primer pairs were typed in 12 unrelated chickens and also tested on Japanese quail and helmeted guinea fowl deoxyribonucleic acid (DNA). Polymorphism was observed in 23 (82.1%) of the markers and the average number of alleles per locus was 2.9 while the mean heterozygosity was 0.19. Eleven (39.3%) of the chicken markers cross-reacted with Japanese quail DNA and 2 (7.1%) with helmeted guinea fowl DNA. The cross-reactive markers described would serve as useful resources for comparative genetic mapping in poultry species belonging to the order Galliformes.