The aim of this present study was to evaluate the formation of micro-cracks in root dentin caused by root canal instrumentation with four different Ni-Ti-Systems. Sixty human single-rooted teeth with straight root canals and mature apices were randomly assigned to four groups (n=15). Preparation of access cavities of the experimental teeth was followed by coronal reduction to 15 mm, resulting in a working length of 14 mm. Consequently, gradual preparation of root canals with assigned systems was carried out. The following four NiTi-systems were used: GT-Series X, Reciproc, Mtwo and S-ApeX. Prior to instrumentation and after each file, microphotograps of the roots were taken for the mesial and distal aspect. Micro-cracks were identified and micro-crack length were measured and cumulated. Each of the four instruments tested induced formation of micro-cracks in root dentin, albeit to varying degrees. The crack growth from the initial to the next following file was significant (p < 0.05). The highest total crack length was measured in teeth treated with the S-ApeX-system, whereas the GT-Series X-system induced lowest total crack length, but the differences between both in-strument systems were not significant (apical: p=0.64, middle: p=0.183, coronal: p=0.298). With regard to the time required for preparation Reciproc and GT-Series X were significantly faster than Mtwo and S-ApeX.