The polychaete genus Laeonereis (Annelida, Nereididae) occurs over a broad geographic range and extends nearly across the entire Atlantic coast of America, from the USA to Uruguay. Despite the research efforts to clarify its diversity and systematics, mostly by morphological and ecological evidence, there is still uncertainty, mainly concerning the species Laeonereis culveri, which constitutes an old and notorious case of taxonomic ambiguity. Here, we revised the molecular diversity and distribution of Laeonereis species based on a multi-locus approach, including DNA sequence analyses of partial segments of the cytochrome c oxidase subunit I (COI), 16S rRNA, and 28S rRNA genes. We examined Laeonereis specimens collected from 26 sites along the American Atlantic coast from Massachusetts (USA) to Mar del Plata (Argentina). Although no comprehensive morphological examination was performed between different populations, the COI barcodes revealed seven highly divergent MOTUs, with a mean K2P genetic distance of 16.9% (from 6.8% to 21.9%), which was confirmed through four clustering algorithms. All MOTUs were geographically segregated, except for MOTUs 6 and 7 from southeastern Brazil, which presented partially overlapping ranges between Rio de Janeiro and São Paulo coast. Sequence data obtained from 16S rRNA and 28S rRNA markers supported the same MOTU delimitation and geographic segregation as those of COI, providing further evidence for the existence of seven deeply divergent lineages within the genus. The extent of genetic divergence between MOTUs observed in our study fits comfortably within the range reported for species of polychaetes, including Nereididae, thus providing a strong indication that they might constitute separate species. These results may therefore pave the way for integrative taxonomic studies, aiming to clarify the taxonomic status of the Laeonereis MOTUs herein reported.