Background: Isoberlinia (Craib and Stapf) is a genus with high economic and pharmacological values.Aim: This study aimed at establishing the morphological, anatomical and molecular characterisation of the leaves of I. doka and I. tomentosa, which were conducted for proper authentication.Setting: The leaves of I. doka and I. tomentosa were obtained from Shika, kaduna State, Nigeria.Method: Morphological and anatomical characters were determined according to standard procedures, while molecular identifications were performed using ribulose-1,5-bisphosphate carboxylase (rbcl) gene and internal transcribed spacer (ITS) DNA barcode’s region.Result: Morphological studies revealed similar features for both species except for the shiny leaves of I. doka and rough abaxial surfaces of I. tomentosa because of the presence of trichomes. Variations were observed in their epidermal features, stomatal index, stomata frequency, presence or absence of trichomes, trichomes frequency and their quantitative anatomical features. The quantity and quality of DNA measured at A260/280 ratio using nanodrop spectrophotometer were 29.1 ng/μL and 1.74 ng/μL for I. doka, respectively, while the I. tomentosa concentration and purity were 71.1 ng/μL and 1.85 ng/μL, respectively. Agarose gel electrophoresis revealed two DNA bands with 700 bp (rbcl) and 600 bp (ITS). The sequence analysis revealed maximum identity with National Centre for Biotechnology Information (NCBI) GeneBank Isoberlinia species. Evolutionary analysis supported the monophyletic origin of the genus Isoberlinia. The morphological and anatomical characters of I. doka and I. tomentosa leaves have provided a significant taxonomy tool for proper authentication of this plant.Conclusion: The findings ascertained that ITS and rbcl served as an improved and efficient tool for species identification of these studied species and could serve as potential DNA barcodes for these taxa.Contribution: This article suggests that further studies the on screening of these plants, for various pharmacological potentials, might be useful for new drug development.