PBac{UAS-nBitbow.1.0} is a multicolor live imaging lineage-tracing tool. The 'nBitbow' cassette consists of multiple fluorescent protein (FP) coding units, each of which is tagged at the N-terminal end with human histone H2B (hH2B) to ensure nuclear localization, with each of these fusion proteins flanked by a pair of FLP recombinase target sites that will recombine with each other but which are incompatible with all the other pairs of FLP recombinase target sites in the transgene. UAS regulatory sequences are positioned upstream of each FP coding unit, although in the absence of FLP recombinase, no FP expression is expected as each coding sequence is initially in reverse orientation. In the presence of FLP recombinase each FP coding unit can stochastically and independently flip orientation into a forward or reverse expression state, resulting in a number of possible color combinations. PBac{UAS-nBitbow.1.0} contains five fluorescent protein coding units, encoding hH2B-tagged mAmetrine, mTFP1, mNeonGreen, mKO2 and tdKatushka2 (flanked by a pair of FRT3, FRT14, FRT545, FRT13 and FRT4 sites respectively), and can thus produce 31 different fluorescent color combinations. When combined with statistical methods, the properties of PBac{UAS-nBitbow.1.0} can be used to quantify the lineage relationship of any two cells.