For over a century, Lepidosiren paradoxa has been recognized as the single lungfish species in South America. Previous studies have failed to identify morphological differences in lungfish populations inhabiting the Amazon and Paraná-Paraguay River basins. Here, we report molecular evidence of cryptic speciation in South American lungfish. Our analysis of mitochondrial gene sequences from 56 L. paradoxa individuals collected in the Amazon and Paraná-Paraguay river systems revealed that genetic distances between individuals from different basins were on average 20 times greater than those obtained for individuals that occur in the same basin. Haplotype network did not uncover shared haplotypes between basins. Additionally, the species delimitation analysis showed that the posterior probability for the existence of distinct species is highly significant (PP = 0.99961). The phylogenetic analyses (BI and ML) recovered individuals from distinctive basins in reciprocally monophyletic clades. Our divergence time estimate suggests that the Lepidosiren in the Amazon basin are seven million years split from those that occur in the Paraná-Paraguay basin. On the other hand, in agreement with previous reports, our morphological comparison of 20 specimens from the Amazon and 14 specimens from the Paraná-Paraguay basins revealed no distinctive morphological features. The combined molecular and morphological results suggest that these two forms may represent cryptic species.