DNA barcoding was used to investigate the phylogenetic delimitations of Eisenia sp. populations used in ecotoxicological research in South Africa. A total of three focal groups (used in published works) and two non-focal groups were assessed. These focal groups, including two laboratory cultures and one field population, have been referred to as Eisenia fetida in the literature. A previous molecular study had already helped to establish that one of the two laboratory groups was a population of E. fetida's sister species E. andrei. In the present contribution, analyses of the COI gene revealed that the taxonomy of the remaining laboratory and field populations had also been incorrectly assigned since all the generated sequences grouped unequivocally with published sequences of E. andrei. Very high sequence divergence (>25% K2P) found within E. andrei could signal the occurrence of hitherto undescribed cryptic species. These findings are discussed with an emphasis on the possible consequences of using poorly identified earthworms or specimens with high molecular divergence in ecotoxicological bioassays. It is not clear whether unbeknownst to the researchers, the use of cryptic species in bioassays could jeopardise the quality of ecotoxicological investigations. Early evidence suggests that cryptic oligochaete species may respond differently to metal toxicity. The need for comparative ecotoxicological studies between E. andrei and E. fetida is also evidenced, especially in the light of recent numerous reports of cryptic oligochaete species. Ecotoxicologists are consequently encouraged, whenever possible, to make use of available genomic technologies to screen their laboratory stocks and available field populations for any molecular distinctiveness.