The genus Chaetostoma is a species-rich group of armored catfishes that inhabits rivers and streams in montane forests of South America. Most species of Chaetostoma have been described based on only one or a few specimens from a single river system. Our aim was to determine if the lifting of the Fitzcarrald Arch, 4 Ma, triggered speciation between the disjunct populations of Chaetostoma lineopunctatum in two isolated river drainages: the Río Ucayali and the Río Madre de Dios. We addressed this question by quantifying morphological and molecular divergence among samples of C. lineopunctatum from four river systems, three of which drain into the Río Ucayali drainage, including the type locality for the species, and one river system which drains into the Río Madre de Dios drainage. Morphological divergence, based on measurements, was statistically significant in the MANOVAs for the samples grouped by locality (P < 0.025) and by river system (P < 0.025). However, neighbor-joining trees of specimens grouped by locality (based on Mahalanobis distances) did not show group patterns according to river system. Morphologically, the specimens can be discriminated by river system with up to 88.7% confidence based on size-free discriminant analysis. Furthermore, the divergence values among haplotypes from the Río Ucayali and the Río Madre de Dios drainages, based on partial cytochrome b sequences (801 bp), ranged from 1.1% to 1.5%, which overlaps the 1.2% divergence between haplotypes from the Río Ucayali drainage. Based on the available morphological and molecular data, we conclude that there are not significant differences among populations of C. lineopunctatum from the Río Ucayali and the Río Madre de Dios drainages, and thus the Fitzcarrald Arch uplift did not cause speciation in this taxon. Alternatively, we suggest that isochronous tectonic events prior to the uplift of the Fitzcarrald Arch could have triggered genetic isolation of populations of C. lineopunctatum.