The Nile tilapia, Oreochromis nilotiucs, is a species of prime importance in local and international aquaculture. It has been receiving an increasing attention in genetic markers development for improving aquaculture. The current study assayed the genetic diversity and population structureof the Nile tilapia in three populations in Egypt (Kafr El-Sheikh, El-Fayoum, and El-Dakahlia). Amplified fragment length polymorphism (AFLP) markers were developed, applying two restriction enzymes (MseI and EcoRI) in three primer combinations (MM1, MM2, MM3). Samples from El-Fayoum exhibited higher genetic richness than El- Dakahlia and Kafr El-Sheikh. Among the studied markers, MM2 showed a higher allele frequency in males compared to females of all locations combined. Furthermore, the analysis of molecular variance (AMOVA) indicated higher genetic variance within locations, as well as within males, and females, rather than among them. Using association genetics between AFLP makers and body weight traits, 22 significant associations were detected after false discovery rate correction (FDR) at P< 0.05. Phenotypic variance (R²) explained by each marker was moderate and ranged between 13.2% for the MM1 (M126, associated with body length) and 30.7% for MM3 (M459, associated with body weight). The current study showed that El-Fayoum population was the most diverse compared to Kafr El-Sheikh and El-Dakahlia populations, with lesser aquaculture activity, calling for efforts to diversify Kafr El-Sheikh and El-Dakahlia populations. The significant marker-trait associations identified in the current study would contribute to the acceleration of tilapia breeding programs.