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Macadamia nuts are known globally for their high quality and economic value. Global macadamia commercial nut production amounts to 60,000 metric tonnes and is increasing steadily. South Africa is the leading producer with 29% of worldwide kernel production. Commercial macadamia germplasm was originally selected from a small genepool (mainly Macadamia integrifolia species) from a limited geographic distribution in Australia. These accessions were subsequently bred, cloned and exported across the world to start local macadamia industries. The South African macadamia industry was established with pre-commercial and commercial macadamia from different parts of the world, and local selections were also performed. Many of these accessions have unique genetic compositions that have not been characterized yet. We used 13 nuclear microsatellite markers to study the genetic diversity and structure of macadamia germplasm cultivated in South Africa. We compared four groups of accessions including 31 originating from the Hawaiian Agricultural Experimental Station (HAES), 19 from Australia (AUS), two from California and one from Israel (OTH), 31 from South Africa's locally selected accessions (SA) and 26 from two local Farmers (FARM). We used STRUCTURE, PCoA and neighbour-joining phylogenetic analyses to show that the South African selected accessions include diverse hybrid genotypes with strong Macadamia tetraphylla composition, unlike the Hawaiian commercially released and Australian representative collections that mostly have M. integrifolia or hybrid composition. Our results suggest that the South African selections represent a unique and diverse set of germplasm for future macadamia improvement efforts that will benefit from genomic breeding technologies. [ABSTRACT FROM AUTHOR]