The DNA barcodes are generally interpreted using distance‐based and character‐based methods. The former uses clustering of comparable groups, based on the relative genetic distance, while the latter is based on the presence or absence of discrete nucleotide substitutions. The distance‐based approach has a limitation in defining a universal species boundary across the taxa since the rate of mtDNA evolution is not constant throughout the taxa. However, character‐based approach more accurately defines this using a unique set of nucleotide characters. The character‐based analysis of full‐length barcode has some inherent limitations, like sequencing of the full‐length barcode, use of a sparse data matrix and lack of a uniform diagnostic position for each group. A short continuous stretch of a fragment can be used to resolve the limitations. Here, we observe that a 154 bp fragment, from the transversion rich domain of 1367 COI barcode sequences can successfully delimit species in the three most diverse orders of freshwater fishes. This fragment is used to design species‐specific barcode motifs for 109 species by the character‐based method, which successfully identifies the correct species using a pattern‐matching program. The motifs also correctly identify geographically isolated population of the Cypriniformes species. Further, this region is validated as a species‐specific mini‐barcode for freshwater fishes by successful PCR amplification and sequencing of the motif (154 bp) using the designed primers. We anticipate that use of such motifs will enhance the diagnostic power of DNA barcode, and the mini‐barcode approach will greatly benefit the field‐based system of rapid species identification. This article is protected by copyright. All rights reserved.