Deltas are dynamic and productive systems of enormous ecological significance, encompassing unique and biologically diverse wetland habitats. Here, we present the first data on the molecular diversity of the fish fauna of the Parnaíba Delta, the largest deltaic formation of the Americas. Partial sequences (626 bp) of the mitochondrial COI gene (Cytochrome c oxidase subunit I) were used to barcode 402 individuals, representing 128 species, belonging to 98 genera, 57 families, 17 orders and two classes. The most abundant orders were the Perciformes, Siluriformes, Gobiiformes, and Pleuronectiformes. The Neighbor-Joining (NJ), Bayesian Inference (BI), and BIN analyses produced 103 molecular clusters, while the Automatic Barcode Gap Discovery (ABGD) and Maximum Likelihood (ML) approaches revealed 102 clusters. The mean conspecific, congeneric and confamilial genetic distances were 0.33%, 14.37%, and 18.60%, respectively. Intraspecific divergence ranged from 0.0% to 1.4%, and all species presented barcode gaps, with the exception of two clusters of Cathorops spixii (OTU 96 and OTU 103), which were separated by a low interspecific distance (1.2%), which overlaps the maximum intraspecific genetic distance (1.4%). The barcode data provide new insights into the fish diversity of the Parnaíba Delta, which will be important for the development of further research on this fauna.