Astyanax bimaculatus are small characids known as piabas or lambaris that form a complex encompassing 18 species, including cryptic species. The present study aimed to use DNA barcode to analyze populations of A. bimaculatus found in Maranhão hydrographic basins, comparing molecular diversity indices between populations from the other Brazilian basins. The results revealed the formation of 32 haplotypes (h = 0.9289; π = 0.0523). Seven haplogroups were formed with intrapopulation genetic distance ranging from 0 to 2%. The Maranhão populations of the Western Northeast Atlantic Region basins separated from the other analyzed basins, corroborating with the groups generated in BAPS and with the Bayesian Inference tree. The occurrence of exclusive OTUs for the Maranhão populations of the Western Northeast Atlantic Region was confirmed through delimitation models. Thus, the data from this study provide information on the genetic diversity of the A. bimaculatus complex with the detection of a different lineage for the State of Maranhão, contributing to the understanding of the group's systematics.