The ergasilid copepod Acusicola margulisae n. sp. is described based on material from three species of cichlid, Amphilophus citrinellus (Günther), Parachromis managuensis (Günther), and Oreochromis sp., and from the poecilid Poecilia mexicana (Steindachner), in the crater Lake Asososca León, Nicaragua. This constitutes the 15th species described in the genus Acusicola Cressey, 1970. The new species differs from all its congeners by the relatively longer first endopodal segment of leg 1, and the size and number of setae on second endopodal segment of leg 1. We provide the first gene sequence for a species of Acusicola. To examine the intraspecific genetic variation of the new species collected from different host species, sequences of the mitochondrial barcode region cox1 were generated. In addition, partial regions of the 18S and 28S ribosomal RNA genes were sequenced and used to infer the phylogenetic relationships of the genus Acusicola within the family Ergasilidae Burmeister, 1835. The phylogenetic trees yielded the isolates of Acusicola margulisae n. sp. as a reciprocally monophyletic lineage, and as the sister taxa of five genera of ergasilid copepods. The genus Ergasilus von Nordmann, 1832 was recovered as a paraphyletic group. These analyses indicate that phylogenetic relationships are not yet well resolved and more representative species and genera of the family are required to provide a robust classification of this highly diverse group of copepods.