Near-infrared fluorescent proteins (FPs) are in high demand for in vivo imaging. We developed four spectrally distinct near-infrared FPs--iRFP670, iRFP682, iRFP702 and iRFP720--from bacterial phytochromes. iRFPs exhibit high brightness in mammalian cells and tissues and are suitable for long-term studies. iRFP670 and iRFP720 enable two-color imaging with standard approaches in living cells and mice. The four new iRFPs and the previously engineered iRFP713 allow multicolor imaging with spectral unmixing in living mice.