The medicinally and economically important tea plant of India lacks a report on barcode study. Thus, we aimed to establish the DNA barcode of some elite tea clones of Darjeeling and Dooars along with the study of variation within the chloroplast region. A thorough investigation of 29 tea clones based on the matK (maturase K) gene has been carried out in our study. The laid objectives were fulfilled following DNA isolation, purification, amplification of the matK region, and sequencing. The sequences were further analyzed using BLAST analysis and phylogenetic tree construction along with the study of the aligned consensus region among all the clones. A BLAST search of NCBI revealed 24 clones to share 100% identity with Camellia sinensis. The remaining 5 clones showed 99.29-99.89% identity with Camellia sinensis. However, clones such as 11125 and 11126 showed a higher percentage of similarity, that is, 99.87% and 99.57% with other species of Camellia when compared, respectively, to 99.61% and 99.29% with Camellia sinensis. The relatedness to other Camellia species was also evident from the distinct cluster in the phylogenetic tree. This study reports a total of 14 variable sites within the matK region where the high consensus region revealed a total of nine variable sites and the low consensus region revealed a total of five variable sites. Therefore, this study is the first report of barcode analysis of Indian tea clones, wherein we successfully utilized the single locus matK gene to study variation within the chloroplast region and also conclude that the matK region is not 100% conserved with the same species of Camellia.