© 2017 The Author(s). Electronic properties of two-dimensional (2D) MoS2semiconductors can be modulated by introducing specific defects. One important type of defect in 2D layered materials is known as rotational stacking fault (RSF), but the coexistence of multiple RSFs with different rotational angles was not directly observed in freestanding 2D MoS2before. In this report, we demonstrate the coexistence of three RSFs with three different rotational angles in a freestanding bilayer MoS2sheet as directly observed using an aberration-corrected transmission electron microscope (TEM). Our analyses show that these RSFs originate from cracks and dislocations within the bilayer MoS2. First-principles calculations indicate that RSFs with different rotational angles change the electronic structures of bilayer MoS2and produce two new symmetries in their bandgaps and offset crystal momentums. Therefore, employing RSFs and their coexistence is a promising route in defect engineering of MoS2to fabricate suitable devices for electronics, optoelectronics, and energy conversion.