Genetic variability of Trichomycterus from the region of Torotoro (Bolivia, Upper Amazon), distributed in the same watershed where the habitat is structured by waterfalls, canyons and a cave, was studied by allozyme (twelve putative loci) and RFLP-mtDNA (DLoop and cytochrome b) analyses. Alloenzymatic variation studied by Correspondence Analysis and Maximum Likelihood Analysis revealed a four-group structure, which was largely congruent with the distribution of the 14 mtDNA haplotypes. Two of these four clusters (I and II) were differentiated by two diagnostic loci (IDH and G3PDH), two semi-diagnostic loci (PGM and 6PGDH) and consequently a very high F(st )value (estimator theta = 0.77). Therefore, clusters I and II are reproductively isolated. The distribution limit of these two (sibling) species does not correspond to those of the morphological species of Trichomycterus identified in this region: the epigean T. cf. barbouri and the hypogean T. chaberti. However, hypogean fish exhibited two mtDNA haplotypes, a private one and another shared with the epigean Trichomycterus from upstream reaches.