A study was carried out to evaluate helmeted guinea fowl (Numida meleagris galeata pallas) in Nigeria using 31 set of microsatellite marker, a total of 200 randomly sampled indigenous guinea fowl strains namely; lavender (ash), gray breasted (white breasted), pearl, black and broiler guinea fowl was used for the study with a sample of forty (40) animals per strain. the Result revealed that the markers were lowly (PIC<0.5) polymorphic in White, Ash and highly (PIC>0.5) polymorphic in Broiler, Black and Pearl guinea fowl. The mean observed heterozygosity (0.102), (0.012), (0.042) is lower than the mean expected heterozygosity (0.591), (0.065), (0.126) in White breasted, Ash and Broiler guinea fowl respectively, with the exception of black and pearl guinea fowl with mean observed heterozygosity (0.101), (0.022) being greater than the mean expected heterozygosity (-0.149), (-0.063). All the populations were in equilibrium, with the exception of AB819301 in black guinea fowl loci that deviated (p<0.01) from Hardy-Weinberg Equilibrium (HWE). The shortest distance (0.4472) was observed between black and white breasted guinea fowl while the distance between Pearl-White breasted (0.6325) and Pearl-Ash (0.6325) were the same. The highest distance (1.0) was observed between broiler guinea fowl and white breasted. The AMOVA showed that the estimated variation (variance) is 6.70 and 0.00 with percentage variation of 100% among population and 0.00 within population respectively. It was also observed that in PC1, pearl (0.57) has the highest positive value followed by broiler guinea fowl (0.388) and then ash (0.057) having the lowest value of eigen vector While Black (0.241) has the highest eigen vector in PC2 followed by Broiler guinea fowl (0.205) and highest negative value of -0.220 attributed by Ash and Pearl followed by -0.007. in PC3 Black, Broiler guinea fowl and White Breasted present a zero eigen vector values of 0.00 with ash having the highest value (0.281) followed by pearl presenting the ix negative (-0.220) value. These 3 PC's account for 97.715% as total variance of which 19.320% was contributed by PC1, while PC2 and PC3 contributed 32.710% and 40.485% respectively. All the eigen values contributed by all the PC'S were below 1.00 finally a distinct cluster was presented by broiler guinea fowl while black and white breasted tends to clustered together in the 1st quadrant. However, Ash and Pearl presented very closer cluster in the 3rd quadrant. Conclusively, the result revealed that the markers were lowly (PIC<0.5) polymorphic in White, Ash and highly (PIC>0.5) polymorphic in Broiler, Black and Pearl guinea fowl indicated a moderate to high gene diversity among the studied markers, the population points towards out breeding with more heterozygote in the studied population and the increase in heterozygosity in the studied population is a good indication for genetic variance at polymorphic sites, rich genetic diversity and selection potential and consistent cluster was formed between the strains which may help to quantify the degree of relationships between guinea fowl strains and provide information of closeness and evolutionary history.