Background: Oligochaetes are valuable bioindicators of the quality of watercourse and lake sediments. The morphological identification of aquatic oligochaetes is difficult, prompting the development of new molecular oligochaete indices based on DNA barcoding and Next-generation sequencing of sorted specimens. In general, the samples for DNA barcoding are fixed in absolute ethanol. However, in the case of aquatic oligochaetes, this medium is not appropriate as it can induce a modification of specimen abundances and of the composition of communities. Therefore, we investigated the possibility to amplify and sequence aquatic oligochaetes fixed in formalin for a short time. We performed guanidine extraction and polymerase chain reaction (PCR) amplification/sequencing of the cytochrome c oxydase I (COI) gene on tissue fragments fixed in formalin for different periods of time (from 1 h to 1 week) and in ethanol. Results: The large majority of aquatic oligochaete specimens fixed in formalin for up to 1 week could be successfully amplified and all obtained sequences were of high quality. The amplification and sequencing success rate of formalin-fixed samples and ethanol-fixed samples was similar. These results suggest that formalin fixation of aquatic oligochaete tissues for a short time does not cause serious damages to DNA and inhibit PCR amplification. Conclusion: The possibility to fix aquatic oligochaetes with formalin before genetic analyses is very promising for diversity monitoring, for construction of a comprehensive DNA barcode library and for development of an index based on Next-generation sequencing analysis of samples composed of sorted specimens.