In the present study, the DNA barcoding of the nematode parasite infecting Ompok bimaculatus and Nemacheilus anguilla fish species was carried out in Barvi Reservoir, Maharashtra. To ascertain the taxonomic status of these nematode parasites, an 18S gene marker was used. Accurate identification of fish parasites is essential to formulate preventive strategies and to study host–environment relations. The present study did barcoding of the nematode parasites of the fishes caught from the Barvi Reservoir using the nuclear 18S rDNA (SSU) sequence. The nuclear 18S rDNA (SSU) was amplified into two overlapping amplicons and sequenced to identify the species based on the sequence similarity with the NCBI GenBank database. The present study sequences (both fragments) showed 98% similarity with the species of Eustrongylides. The average genetic distance value between the present study sample and species of Eustrongylides was 0.003. In the phylogenetic tree also, the sequence was clustered with the species of Eustrongylides with significant bootstrap values. The present study identified the nematode parasite of the fish caught from the Barvi Reservoir, as species of Eustrongylides. The species‐level identification could not be possible due to the insufficient/lack of reference sequences in the database. It indicates the knowledge gap concerning the species‐specific molecular markers for nematode parasites of the fish.