Background: The family Aegisthidae is known as typical component of deep-sea hyperbenthic waters that gradually colonized other marine environments. The phylogenetic relationships within this family have been examined here including hyperbenthic, planktonic, benthic forms and two associated Aegisthidae species. Results: Ninety four specimens belong to 14 genera were studied using 18S and 28S rRNA and COI mtDNA. Bayesian analysis supports the monophyly of 10 genera whereas Andromastax, Jamstecia, Nudivorax and Aegisthus revealed to be paraphyletic. The first offshoot of the phylogenetic tree is a clade consists of the undescribed genus Aegisthidae gen.1 sister to the two monophyletic genera Cerviniella and Hase, whereas the other Cerviniinae members (represented by Cervinia and Expansicervinia) assemble a monophylum, sister to the hyperbenthic and planktonic aegisthid genera, resulting in the paraphyly of the subfamily Cerviniinae. Hence, we defined the new subfamily Cerviniellinae subfam. nov. encompassing the three benthic genera Cerviniella, Hase and Eucanuella. The subfamily Cerviniinae has been redefined to include Cervinia, Expansicervinia and Paracerviniella. Members of the subfamily Pontostratiotinae were clustered into two clades, one consists of the genus Stratiopontotes sister to an undescribed genus + Cerviniopsis and Siphonis. The second contains Pontostratiotes sister to the members of the planktonic subfamily Aegisthinae, resulting in the paraphyly of the Pontostratiotinae. Therefore, the Pontostratiotinae has been redefined to include only members of the genus Pontostratiotes; whereas the subfamily Cerviniopseinae has been re-erected and redefined containing Stratiopontotes, Cerviniopsis, Siphonis, Aegisthidae gen. 2, Herdmaniopsis, Hemicervinia and Tonpostratiotes. Within this subfamily, the associated Siphonis clusters as sister to the Cerviniopsis represents an example of convergent evolution in which the possession of a stylet-like mandible and an oral cone reminiscent of the Siphonostomatoida. The planktonic Aegisthus, Andromastax, Jamstecia, Nudivorax and Scabrantenna confirm the monophylom Aegisthinae, sister to the Pontostratiotinae. Conclusions: Our DNA based phylogeny reveals the deep-sea origin of Aegisthidae by placing benthic Aegisthidae gen.1 and Cerviniellinae subfam. nov. as the most basal lineages. Secondary adaptations to hyperbenthic and planktonic realms, as well as associated lifestyle were discovered here by the derived positions of Pontostratiotinae, Aegisthinae and Siphonis respectively.