Nile tilapia (Oreochromis niloticus L.) is a worldwide significant aquaculture species rapidly gaining rank as a farmed commodity. Six fish specimens (three males and three females) of Nile tilapiawere collected from Kafr El-Sheikh Egypt (31.3^oN and 30.93^oE). Fluorescent Amplified Fragment Length Polymorphism (AFLP) analysis was applied to qualitatively discriminate sex-dependent genomes, was successfully able to determine sex-linked marker. Principal Coordinate Analysis (PCA) identified sex-linked marker easily. PCR amplification was obtained using three pairs of primers. Total number of scored bands detected the specimens   was 1243 as 129, 154, 291, 148, 165 and 356 bands for the three primer pairs, respectively. All bands ranged between 50 to 542 bp including 919 polymorphic. Percentage of polymorphic loci was 73.93%. The PCA test revealed different distribution of male and female samples according to the degree of similarity. The difference in PCA revealed genetic variation among the studied samples. Such observations reflect the success of the F- AFLP markers. A PCA variable showed clustering in four quadrants variables for female and male samples in different quadrants.