Tilapia are amongst the most important farmed fish species worldwide, and are fundamental for the food security of many developing countries. Several genetically improved Nile tilapia (Oreochromis niloticus) strains exist, such as the iconic Genetically Improved Farmed Tilapia (GIFT), and breeding programmes typically follow classical pedigree-based selection. The use of genome-wide single-nucleotide polymorphism (SNP) data can enable an understanding of the genetic architecture of economically important traits and the acceleration of genetic gain via genomic selection. Due to the global importance and diversity of Nile tilapia, an open access SNP array with known utility across multiple tilapia strains would be beneficial for aquaculture research and production. In the current study, a ~65K SNP array was designed based on SNPs discovered from whole-genome sequence data from a GIFT breeding nucleus population and the overlap with SNP datasets from several other farmed and wild Nile tilapia strains. The SNP array was applied to clearly distinguish between different tilapia populations across Asia and Africa, with at least ~30,000 SNPs segregating in each of the diverse population samples tested. It is anticipated that this SNP array will be an enabling tool for population genetics and tilapia breeding research, facilitating consistency and comparison of results across studies.