Sprentascaris mahnerti (Nematoda: Raphidascarididae) collected from Loricariichthys labialis (Siluriformes: Loricariidae) in the Pantanal wetlands, State of Mato Grosso do Sul (Brazil), was redescribed using light and scanning electron microscopy (SEM), and genetically characterised along with two other raphidascaridids: Raphidascaroides brasiliensis and Ro. moraveci. Due to the systematic discussion regarding Raphidascaris and Sprentascaris, as well as the poor knowledge about the phylogenetic relationships within Raphidascarididae, phylogenies were reconstructed based on partial sequences of the 18S and 28S nuclear rRNA gene, the nuclear ITS1-5.8S-ITS2 and the cytochrome c oxidase subunit I (cox1) mtDNA. Morphological study of S. mahnerti, confirmed some previously described features, revealed new characteristics and permitted to elucidate some inconsistencies noted in the literature. Morphological and genetic characterisation of S. mahnerti supported its validity. Phylogenetic reconstructions supported the monophyly of Sprentascaris, which has three pairs of interlabial conspicuous cuticular projections as a synapomorphy. The relationships among several lineages of raphidascaridids were unsolved, albeit Goezia and Ichthyascaris formed well-supported monophyletic assemblages, in which the first included species with no relations regarding the habitat of hosts and the geographic origin. The present findings represent one more step towards the understanding of the interrelationships of raphidascaridid nematodes. In this sense, Sprentascaris should be considered valid as an independent lineage from Raphidascaris.