We show that the lack of inversion symmetry in monolayer MoS2 allows strong optical second harmonic generation. The second harmonic of an 810-nm pulse is generated in a mechanically exfoliated monolayer, with a nonlinear susceptibility on the order of 10−7 m/V. The susceptibility reduces by a factor of seven in trilayers, and by about two orders ofmagnitude in even layers. A proof-of-principle second harmonicmicroscopymeasurement is performed on samples grown by chemical vapor deposition, which illustrates potential applications of this effect in the fast and noninvasive detection of crystalline orientation, thickness uniformity, layer stacking, and single-crystal domain size of atomically thin films of MoS2 and similar materials.