The aim of this study was to evaluate the effect of glide path preparation on the surface topography changes of WaveOne and OneShape single files using atomic force microscopy analysis. Two commercially available brands of single file systems were analyzed, including 15 specimens/brand as following: Group I; WaveOne 0.08/25 (Dentsply Maillefer, Ballaigues, Switzerland) and Group II; OneShape 0.06/25 (Micro Mega, Besancon, France). In each group, 3 new files were subjected to AFM analysis as received from manufacturer, and 12 files were analyzed after preparation of root canals. A total of 20 human maxillary molar teeth were selected. In each group, 12 files were divided into 4 subgroups according to instruments that were used in one or three root canals with or without a previous glide path (n=3). After preparation the surface analysis of single files was performed on 11 different points located along a 3-mm section starting at the tip of each file using AFM. Root mean square (RMS) values were recorded to investigate the surface features of endodontic files. The differences between RMS values were tested by one-way ANOVA. There was no statistically significant difference between the groups (p>0.05). Creation of a glide path did not affect the surface topography of single file systems. SCANNING 36:608-613, 2014. (c) 2014 Wiley Periodicals, Inc.