The growing demand on Oreochromis niloticus as a popular food increased interest in its aquaculture. This necessitated proper differentiation of O. niloticus from different localities to help in choosing the best specimens for brood stock production for Nile tilapia farming. Discriminant analysis was used to quantify morphometric measurements and meristic counts of O. niloticus from Kosti, Sinnar, Khashm El Girba and Al Sabloga area. Function 1 of Discriminant analysis separated O. niloticus sample from Kosti, Sinnar, and Khashm El Girba samples. Function 3 separated Kosti samples from other areas. Function 3 separated Kosti samples from other areas. Function 2 separated Kosti and Khashm El Girba samples from Sinnar and Al Sabloga samples. Discriminant analysis showed high degree of purity of O. niloticus with an overall average of 85.1%, this because the specimens from one location sharing characters with other from different location are very few. Wilks lambda analysis showed extremely highly significant difference (p<0.000) between the three functions. This analysis selected 12 morphometric characters (PP, HL, HW, IOW, AFB, CD, LAD, SNL, CPL, ED, CPD and PRP) to be used with high accuracy to discriminate between O. niloticus from the four sampling locations. Leave-one-out cross validation for O. niloticus from four locations by Discriminant analysis using 19 morphometric characters and 6 meristic counts confirmed the identity of the species.