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We describe the optimization and validation of six DNA isolation protocols from fresh leaves of the rare tree Acacia pachyceras. The first four protocols employed three commercial kits (Sigma, Nucleospin1, Nucleospin 2, Promega) whereas the remaining two were based on the traditional sodium dodecyl sulfate (SDS) and cetyltrimethylammonium bromide CTAB methods. Each protocol provided significantly different results concerning DNA concentration (p Q30). These protocols will be useful tools for molecular research of Acacia pachyceras and other closely related tree species.