BACKGROUND: A large part of our country's waste is organic matter, so researchers are studying fertilizer production from organic waste in various ways, including compost and vermicompost. On the other hand, glycolipoprotein extract of Eisenia fetida (G-90) has been defined to show numerous biological activities, e.g., anticoagulation, fibrinolysis, and anti-oxidative, etc. OBJECTIVES: The purpose of the present study was to determine phylogenetic relationship of the Iranian isolate of Eisenia fetida (E. fetida) with the other available taxa and to determine the G-90 protein complex for evaluation of its biological activities. METHODS: A piece of 1×1cm clitellum was separated and used for DNA extraction after homogenate preparation (by two different methods). The second internal transcribed spacer of the nuclear ribosomal DNA (rDNA-ITS2) and cytochrome C oxidase subunit 1 of the mitochondrial DNA (mtDNA-COX1) from adult E. fetida were amplified using polymerase chain reaction (PCR). Subsequently, PCR products were sequenced and their phylogenetic relationships were determined. Further-more, The G-90 protein complex was extracted from adult worms and electrophoretic pattern of proteins was obtained by sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE). RESULTS: The electrophoretic pattern of glycolipoprotein G-90, a protein complex, showed 10 bands with molecular weights of 14-130 kDa. ITS2 and COX1 sequences were 516 bp and 277 bp long, respectively. The amplified DNA sequences from both ribosomal and mitochondrial sequences had 88%-99% and 99% similarity to relevant sequences in GenBank, respectively. CONCLUSIONS: mtDNA-COX1 showed no considerable sequence variations as compared to other isolates in Gen-Bank, while rDNA-ITS2 exhibited more variations, in comparison with other isolates, indicating more variations among some of these isolates. Our results revealed that rDNA-ITS2 of our E. fetida was in a separate subclade, showing the greatest similarity with EF534709.1 isolate (99%) provided in GenBank, followed by JX531618.1 (94%), and KU708469.1 (88%). Our COX1 sequence demonstrated the high similarity of 99% when compared with five isolates from GenBank (MH475674.1, MH475673.1, MH475672.1, MH475670.1, and MH475666.1). The G-90 glycolipoprotein showed different proteins that can be assessed for their potential biological activities in medicinal properties