The high-order structure of mitotic chromosomes remains to be fully elucidated. How nucleosomes compact at various structural levels into a condensed mitotic chromosome is unclear. Cryogenic preservation and imaging have been applied for over three decades, keeping biological structures close to the native in vivo state. Despite being extensively utilized, this field is still wide open for mitotic chromosome research. In this review, we focus specifically on cryogenic efforts for determining the mitotic nanoscale chromatin structures. We describe vitrification methods, current status, and applications of advanced cryo-microscopy including future tools required for resolving the native architecture of these fascinating structures that hold the instructions to life.