© The Royal Society of Chemistry 2017. Brighter near-infrared (NIR) fluorescent proteins (FPs) are required for multicolor microscopy and deep-Tissue imaging. Here, we present structural and biochemical analyses of three monomeric, spectrally distinct phytochrome-based NIR FPs, termed miRFPs. The miRFPs are closely related and differ by only a few amino acids, which define their molecular brightness, brightness in mammalian cells, and spectral properties. We have identified the residues responsible for the spectral red-shift, revealed a new chromophore bound simultaneously to two cysteine residues in the PAS and GAF domains in blue-shifted NIR FPs, and uncovered the importance of amino acid residues in the N-Terminus of NIR FPs for their molecular and cellular brightness. The novel chromophore covalently links the N-Terminus of NIR FPs with their C-Terminal GAF domain, forming a topologically closed knot in the structure, and also contributes to the increased brightness. Based on our studies, we suggest a strategy to develop spectrally distinct NIR FPs with enhanced brightness.