The fast development of aquaculture over the past decades has made it the main source of fish protein and led to its integration into the global food system. Mostly originating from inland production systems, aquaculture has emerged as strategy to decrease malnutrition in low-income countries. The Nile tilapia (Oreochromis niloticus) was introduced to Madagascar in the 1950s, and is now produced nationally at various scales. Aquaculture mostly relies on fry harvested from wild populations and grow-out in ponds for decades. It has recently been diversified by the introduction of several fast-growing strains. Little is known how local genetic diversity compares to recently introduced strains, although high and comparable levels of genetic diversity have previously been observed for both wild populations and local stocks. Our study compares DNA barcode genetic diversity among eight farms and several strains belonging to three species sampled. DNA-based lineage delimitation methods were applied and resulted in the detection of six well differentiated and highly divergent lineages. A comparison of DNA barcode records to sequences on the Barcode of Life Data System (BOLD) helped to trace the origin of several of them. Both haplotype and nucleotide diversity indices highlight high levels of mitochondrial genetic diversity, with several local strains displaying higher diversity than recently introduced strains. This allows for multiple options to maintain high levels of genetic diversity in broodstock and provides more options for selective breeding programs.