Analysis of the genetic diversity of tropical trees can contribute to forming active approaches for their conservation and future use. High-quality Deoxyribonucleic acid (DNA) is required for many molecular applications used in the analysis of genetic diversity. The present study standardized efficient DNA isolation and long-term storage protocol using the 268 leaf samples from 8 tropical tree species such as Cullenia exarillata (Malvaceae), Dalbergia latifolia, Dalbergia sissoides, Kingiodendron pinnatum (Fabaceae), Dysoxylum malabaricum (Meliaceae), Mesua ferrea (Calophyllaceae), Myristica malabarica (Myristicaceae) and Tectona grandis (Lamiaceae). The high DNA quality resulted in the present method as Mean A260/280 = 1.90 ± 0.05. The amount of extracted DNA ranged from 23.4 ng/µL (in the mature leaf of Myristica malabarica) to 1922.7 ng/µL (in the young leaf of Dalbergia sissoides), while the yield ranged from 4.7 to 384.5 µg per 100 mg leaf sample. The extracted DNA samples of 8 species stored with 70% ethanol in the pellet form showed less DNA degradation and a maximium storage life of up to one year than those stored with TE buffer and sterile water. The integrity of one year of DNA stored in 70% ethanol was suitable for PCR amplification with RAPD and ISSR markers. The present method also facilitated efficient molecular characterization and validation of tropical tree species.