Knowledge of genetic diversity and barcoding of yam is lacking in Enugu and Ebonyi States of southern Nigeria. Therefore, DNA barcoding was used to facilitate identification and biodiversity studies of yam species from Southern Nigeria. Seventy five yam accessions were collected from Enugu and Ebonyi States, including International Institute of Tropical Agriculture for DNA extraction and amplification using a chloroplast DNA (cpDNA) ribulose-1,5bisphosphate carboxylase (rbcL) marker. There was high level of similarity among the accessions and presence of 534 conserved and 7 variable sites. A transversional mutation of G/T at a consensus position of 335 was identified followed by transitions at 362 (A/G), 368 (A/G), 371 (C/T) and 391 (C/T) within the accessions. Phylogeny resolved the yam accessions into ten major groups with their bootstrap values ranging from 0 -100. Phylogenetic diversity was highest in group X, followed by VII, VI and IX. The inter-group genetic distance based on Kimura 2-parameter model ranged from 0.5000 ± 0.4770 -5.0560 ± 2.5760, while the intra-group had 0.5250 ± 0.5000 -2.0103 ± 1.2579. The mean genetic diversity within the entire population was 0.7970 ± 0.06910. BLAST analysis of total bit score, query coverage, and percentage identity were in the ranges of 411 -1011, 99% -100% and 97% -100%, respectively. However, the rbcL could not resolve the yam accessions well following the comparative assessment of some discrepancies in the detected number of species from phylogenetic groupings, genetic diversity indices and NCBI BLAST hits, thereby, exposing the inefficiency of this marker in dis-How to cite this paper: Ude, G.N., Igwe, et al.criminating the yam accessions. It was demonstrated that rbcL is not an effective marker; therefore, it should not be recommended as a standard-alone marker of choice for DNA barcoding of yam accessions, especially, when accurate identification, discrimination and estimation of genetic diversity of this vital crop are of paramount importance for crop improvement and germplasm conservation.