The relationship between dislocations and mechanical properties of single hemp fibres (Cannabis sativa L. var. Felina) was studied using a microtensile testing setup in a 2-fold approach. In a first investigation the percentage of dislocations was quantified using polarized light microscopy (PLM) prior to microtensile testing of the fibres. In a second approach PLM was used to monitor the dislocations while straining single fibres. The first part of the study comprised 53 hemp fibres with up to 20% of their cell wall consisting of dislocations. For this data set the percentage of dislocations did not affect the mechanical properties. In the second part of the study it was found that dislocations disappeared during tensile testing, and that they did not reappear until several weeks after failure. A strain stiffening effect due to the straightening of the dislocations was not observed. It is possible that the former positions of the dislocations functioned as locations for crack initiation. However, the crack does not propagate transversely all the way trough the dislocation but results in a shear failure between the microfibrils. In rheological studies fibres were strained at constant stress levels, and dislocations that had disappeared did not reappear during that period.