Evaluation of agronomic practices, genetic diversity and population structure of Moringa oleifera (Lam.) is crucial to its sustainable utilization to ensure food and nutritional security. Four agronomic practices of field preparation and soil analysis, regeneration, weeding and pruning, as well as harvesting, were adopted. Genetic diversity and population structure of 20 populations were evaluated using amplified fragment length polymorphism (AFLP) primer pairs (E-ACC/M-CAC and E-ACA/M-CAG). The effects of agronomic practices on leaf quantity production were evaluated using standard procedures. The physico-chemical and morphological data were analyzed using descriptive statistics, while genetic diversity, population structure, dendrogram reconstruction, and Principal Component Analysis (PCA) were analyzed from the AFLP genetic data. Significant effects (P ≤ 0.05) of agronomic practices on the quantity of leaf production were recorded across the accessions. The two primer pairs generated a total of 80 alleles with a mean major allele frequency of 0.0250. Gene diversity and polymorphic information content (PIC) values were high with a mean of 0.98 and 0.974, respectively. The Gst value of 0.0490 indicated that 5 % of the total genetic divergence was among the population while 95 % within the population. Dendrogram reconstruction with genetic distance ranging from 0.87 to 0.96 segregated the accessions into eleven clusters while PCA generated six cluster groups. Field preparation and physico-chemical soil properties, regeneration, weeding and pruning, and stand development, as well as harvesting, influenced the leaf yield and genetic diversity observed in this study. The genetic data revealed that some accessions were clustered along eco-geographical lines while others grouped disparately. Identified potential parent genotypes with valuable and desirable genetic traits can be exploited for commercial, breeding and conservation purposes to ensure sustainable utilization of the species in Nigeria.