Mitofusin1 (Mfn1) mediates outer mitochondrial membrane (OMM) fusion in Opisthokonts. The uncharacterized TM comprises to two helices (namely, the TM1 and TM2) connected by an intermembrane loop. Consistent with previous studies, our results from in silico analyses show that all mitofusins lack N terminal-MTS and the TM may act an internal MTS. We have identified a conserved region in TM domain that is responsible for mitochondrial localization of Mfn1/2. Thus, our results suggest the dual function of TM; in OMM anchoring and signaling Mfn1 to mitochondria. Our study illuminates the underlying role of TM for mitochondrial localization of Mfn1 on one hand and also paves a way for the development of tools for in silico prediction of cellular localization of proteins. •Human Mfn1 contains internal MTS localized in the loop region between the two helices of the conserved transmembrane domain.•Internal MTS is a shared feature of all opisthokont mitofusins.•It facilitates anchoring and targeting of these proteins to the mitochondrial membrane.