Acacia trees are vital to Kenyan ecosystems, contributing to soil enrichment, biodiversity, and wildlife support. They belong to the Fabaceae family and include species like Acacia nilotica and Acacia xanthophloea, which are essential for agroecosystem restoration, land reclamation, and local livelihoods in arid areas. These trees offer resources such as fodder, medicinal products, timber, and honey, with some species also showing potential for new uses, like vegetable tannin from Acacia xanthophloea bark. Recent research using advanced DArTseq technology aims to explore the genetic diversity and population structure of Acacia trees in Kenya, providing crucial data for conservation and sustainable management. Four populations containing seven samples of the ancient Acacia tree were used to investigate genetic diversity through DArTSeq technology. The samples were collected from different locations and DNA was extracted, libraries generated, and sequenced under the Illumina Hiseq 2500 system. The data was then filtered for SilicoDArT and SNP marker calling and generation. The genetic diversity among Acacia samples was low, indicating minimal environmental and human impact on the species. Older tree samples from Kitui and Naivasha exhibited slight differences in unique alleles, contributing to variance within populations. The study revealed that while the DArT platform effectively genotyped indigenous Acacia species, the genetic diversity was low, suggesting potential vulnerability to environmental and genetic challenges. This, high polymorphisms and moderate genetic differentiation indicate that Acacia species have not been significantly impacted by human activities, highlighting their potential for future research and conservation efforts.