"Three methylene spacer" bis(benzimidazolium) derivatives act as a new template for threading dibenzo-24-crown-8 into pseudorotaxanes. In this Article we sought to unveil the difference in the extent of threading of various "three methylene spacer" bis-benzimidazolium moieties based on differences in aromatic methyl substituent positions and anions through the macrocycle dibenzo-24-crown-8. The temperature of the systems were also varied (low temperatures of 240 K and 253 K), when such interwinding of the thread and crown were not detected at room temperature of 308 K. The presence of such threaded complexes was determined based on 1H-NMR initially and finally corroborated by high resolution mass spectrometry and DFT calculations. 2D-NMR experiments ( 1H- 1H-NOESY) proved to be a very important tool in elucidating the interaction present between the components of the pseudorotaxanes. The dethreading/rethreading process was studied. DFT optimized structures suggest lower energy H-bonding orientations and compare the effect of methyl substituents on the axle. © 2012 The Royal Society of Chemistry.