Polarization-resolved second harmonic generation microscopy (PSHG) is becoming an increasingly popular and important technique for differentiating biological tissues based on their disease state, as well as for identifying components related to their ultrastructure that may help in understanding disease progression. In particular, PSHG has shown promise in cancer diagnosis, where PSHG can be used to differentiate the disorganized ultrastructure of collagen from within the extracellular matrix of cancerous tissues from normal tissues. Here, PSHG microscopy techniques and their application in cancer diagnosis over the past 5 years (2020–2024) are described. The PSHG techniques reviewed include polarization-in SHG, a technique where the laser linear polarization is modulated, widefield polarization-in SHG as well as techniques in which the laser polarization is modulated and the polarization state of the outgoing SHG signals is detected, including polarization-in, polarization-out SHG and widefield reduced double Stokes-Mueller SHG. This chapter concludes by describing advances of PSHG to enable the possibility of automated cancer diagnosis and in vivo tissue studies.