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Lake Kolleru is one of the largest freshwater lakes in India and the only RAMSAR site of Andhra Pradesh. Due to several anthropogenic activities, it is currently experiencing an alarming decline in fish biodiversity. This emphasizes an immediate need for initiating research based on molecular tools in addition to traditional methods. The present study was aimed to evaluate the genetic diversity of Kolleru fishes and to compare them with their counter parts across India based on COI sequence data. The analysis was carried out by using 16 COI sequences of Kolleru fishes and 121 sequences of fishes belonging to other parts of India. The COI barcodes clearly distinguished all the fish species with higher inter-specific distance values than intra-specific K2P values. The average genetic distance within species, genus, family, and order was 0.16%, 2.45%, 5.30% and 13.71% respectively. Composition of four nucleotides and GC% at three codon positions were calculated and compared them with other retrieved COI sequences. NJ tree revealed distinct clades showing Kolleru sequences are clustered with other COI sequences of same species with significant bootstrap values. Hence, the COI sequence information is proved to be effective in genetic variation and phylogenetic assessment with respect to conservation of Kolleru fishes. [ABSTRACT FROM AUTHOR]