A recent taxonomic revision of the Neotropical catfish genus Rhamdia (Pisces: Siluriformes: Heptapteridae) reduced a number of described species to synonymy, especially under a broadly circumscribed R. quelen. Evidence is presented here from DNA sequence data, external morphology, and morphometrics that argues for the recognition of R. guatemalensis in Central and northern South America and R. saijaensis and R. cinerascens in the Pacific drainages of Colombia and Ecuador, respectively. The DNA data indicate that all trans-Andean samples form a monophyletic group, within which there are separate clades corresponding to R. laticauda and the synonymized R. guatemalensis, R. saijaensis, and R. cinerascens. The morphometric data substantiate the phylogenetic groupings, and in external morphology, each putative species has diagnostic characters. Rhamdia guatemalensis is characterized by insertion of the adipose fin closer to the dorsal fin than to the caudal fin and presence of a conspicuous lateral longitudinal dark band; R. saijaensis is characterized by a small head with head length 20.8–23.4% of standard length and by lacking a lateral longitudinal band; and R. cinerascens is characterized by a large head with head length 25.8–30.1% of standard length, base of the adipose fin 30.3–33.3% of standard length, outer mental barbels extending to the base of the pectoral rays, and presence of a faint lateral longitudinal band. The external morphological differences and phylogenetic relationships indicate that these groups are both recognizable and represent independent lineages, which argue for their recognition as species.