Three new Acanthodrilinae species from the states of Oaxaca, Tabasco and Chiapas in southern Mexico are described, Diplotrema oaxacana sp. nov., Diplotrema tabascensis sp. nov., and Diplotrema kaxyebensis sp. nov., respectively. D. oaxacana sp. nov. is characterized by the presence of genital setae in segments 8 and 9, a spermathecal ampulla transversally connected to the duct and paired seminal vesicles in 11 and 12. D. tabascensis sp. nov. is separated from all Diplotrema species by genital setae in segment 16. The singleton D. kaxyebensis sp. nov. is characterized by a single pair of seminal vesicles in 12, genital setae in 8 and by a particular arrangement of spermathecal duct-ampulla-diverticulum connections. It is discussed that the neotropical Diplotrema species lineage does probably not belong to the southern hemisphere Diplotrema lineage, which indeed contains the majority of species; its taxonomic separation, however, should wait until more information is obtained on the molecular relationships among neotropical Acanthodrilinae. Finally the topic of describing new earthworm species on singletons is discussed; a decision table is provided to help earthworm taxonomists to decide about this aspect. This paper is dedicated to Dr. Pietro Omodeo for his important contribution to the fields of taxonomy and biogeography of earthworms, on the occasion of his 100th birthday.
Keywords: Annelida, Acanthodrilinae, Mexico, Oaxaca, Chiapas, Tabasco, singletons.