<p>The standard plant DNA barcoding markers viz. ribulose-1,5-bisphosphate Carboxylase/Oxygenase large subunit (<i>rbcL</i>) and maturase K (<i>matK</i>) were used in the identification and discrimination of pteridophyte species. The sequence variations and PCR performance of the studied markers was also assessed. Although we were able to identify all the sampled pteridophytes, both markers showed a varying success rate of amplification. The <i>matK</i> locus (41.4%) had a significantly lower PCR amplification and sequence recovery rate than that of <i>rbcL</i> (87.5%). All the species sampled were well-differentiated by <i>rbcL</i> locus with average genetic divergence based on Kimura 2 parameter (K2P) variability of 5.02% (SE = 0.12) within genera and 14.12% (SE = 0.13) within families. The computed K2P within genera and family for the <i>matK</i> locus was 15.1% (SE = 5) and 37.4% (SE = 0.9), respectively. Cluster analysis using <i>rbcL</i> alone resolved all the pteridophyte samples satisfactorily and exhibited a common congeneric relationship trend. The report reckons the success of <i>rbcL</i> over <i>matK</i> locus in diversity studies of pteridophytes in the Western Ghats and supports the need for new secondary loci to replace <i>matK</i> in a standard 2-locus barcode to supplement <i>rbcL</i>.</p>