Naidinae (former Naididae) is a group of small aquatic clitellate annelids, common worldwide. In this study, we evaluated the phylogenetic status of Naidinae, and examined the phylogenetic relationships within the group. Sequence data from two mitochondrial genes (12S rDNA and 16S rDNA), and one nuclear gene (18S rDNA), were used. Sequences were obtained from 27 naidine species, 24 species from the other tubificid subfamilies, and five outgroup taxa. New sequences (in all 108) as well as GenBank data were used. The data were analysed by parsimony and Bayesian inference. The tree topologies emanating from the different analyses are congruent to a great extent. Naidinae is not found to be monophyletic. The naidine genus Pristina appears to be a derived group within a clade consisting of several genera (Ainudrilus, Epirodrilus, Monopylephorus, and Rhyacodrilus) from another tubificid subfamily, Rhyacodrilinae. These results demonstrate the need for a taxonomic revision: either Ainudrilus, Epirodrilus, Monopylephorus, and Rhyacodrilus should be included within Naidinae, or Pristina should be excluded from this subfamily. Monophyly of four out of six naidine genera represented by more than one species is supported: Chaetogaster, Dero, Paranais, and Pristina, respectively.