The present study was focused on the discovery of single‐nucleotide polymorphisms (SNPs) and measuring genetic variation in three YY‐male and five mixed‐sex Nile Tilapia Oreochromis niloticus strains, which are available in the United States. We performed restriction site‐associated DNA sequencing for SNP discovery and genotyping. Overall, 31,813 SNPs were identified and used to measure both intra‐ and interstrain genetic variation. Mean expected heterozygosity (He) ranged from a low of 0.084 in the Miami YY strain to a high of 0.222 in the Miami mixed‐sex strain. Two of the three YY groups (Miami YY and Fishgen YY Red) had the lowest He values (0.084 and 0.085, respectively) and the second‐ and third‐lowest observed heterozygosity (Ho) values (both Ho = 0.107). The number of private alleles varied from 90 to 3,795 in different strains and tended to increase with increasing He. Inbreeding, as measured by FIS, was found to be low in all populations, ranging from −0.060 ± 0.004 (mean ± SE) in the Fishgen YY Dark strain to 0.04 ± 0.0093 in the GIFT (Genetically Improved Farmed Tilapia) strain; this indicates that the analyzed strains are currently being maintained with approximately random mating of individuals. Genetic differentiation between populations, as measured by pairwise FST, ranged from little differentiation (FST = 0.06 for the Ismailia Canal and GIFT strains) to strong differentiation (FST = 0.32 for the Miami YY and Lake Manzala strains). The discovered SNPs and quantification of genetic variation can be used for parentage determination and high‐throughput pedigree reconstruction as well as for determining the optimal interstrain crosses to achieve heterosis.